There were few early signs of compromise as the most serious bid yet to end three years of bloodshed began, with Syrian Foreign Minister Walid Muallem dubbing the opposition "traitors" and foreign agents.
UN leader Ban Ki-moon had opened the conference with a plea for both sides to set aside their differences in order to save lives.
"After nearly three painful years of conflict and suffering in Syria, today is a day of hope," Ban said.
"You have an enormous opportunity and responsiblity to render a service to the people of Syria."
Meeting for the first time since the start of the conflict in March 2011, the two sides could not be further apart at the "Geneva II" conference in Montreux on the shores of Lake Geneva.
The opposition heads into the conference with a sole aim — toppling President Bashar al-Assad — while the regime says any talk of removing the Syrian leader is a "red line" it will not cross.
Muallem wasted no time firing a broadside at the opposition in his opening speech, which went on long beyond the allotted time of less than ten minutes, forcing Ban to repeatedly ask the foreign minister to stop.
"They (the opposition) claim to represent the Syrian people. If you want to speak in the name of the Syrian people, you should not be traitors to the Syrian people, agents in the pay of enemies of the Syrian people," Muallem said.
Ahmad Jarba, the head of the opposition Syrian National Coalition, called on the regime to "immediately" sign a deal reached at the last peace conference in Geneva setting out "the transfer of powers from Assad, including
for the army and security, to a transition government."
He said the creation of a transition government would be "the preamble to Bashar al-Assad's resignation and his trial alongside all the criminals of his regime."
US Secretary of State John Kerry insisted Assad cannot be part of any transitional government.
"There is no way, not possible in the imagination, that the man who has led the brutal response to his own people could regain legitimacy to govern," Kerry said.
Expectations are very low for a major breakthrough at the conference, but global diplomats gathered here believe that simply bringing the two sides together is a mark of some progress and could be an important first step.
Russian Foreign Minister Sergei Lavrov warned the talks will "not be simple, they will not be quick" but urged both sides to seize a "historic opportunity".
About 40 nations and international groups were gathered in Montreux on Wednesday, but no direct talks are expected until Friday — when opposition and regime delegations will meet in Geneva for negotiations that officials have said could last seven to ten days.
Erupting after the regime cracked down on protests inspired by the Arab Spring, the civil war has claimed more than 130,000 lives and forced millions from the homes.
Recent months have seen the conflict settle into a brutal stalemate — with the death toll rising but neither side making decisive gains.
With no one ready for serious concessions, world powers will be looking for short-term deals to keep the process moving forward, including on localized ceasefires, freer humanitarian access and prisoner exchanges.
Both Washington, which supports the opposition and has called for Assad to step down, and Moscow, the regime's key international supporter and arms provider, will be pushing for progress, but officials have warned the talks will be extremely difficult.
Notably absent will be crucial Assad backer Iran, after Ban reversed a last-minute invitation when the opposition said it would boycott if Tehran took part.
It took months of discussions to convince all sides to participate, with the main opposition group, the Syrian National Coalition, only agreeing at the 11th hour.
That move has seen the Coalition branded as traitors by some in Syria, including Islamist rebels who have often been at the forefront of fighting.
And the biggest bloc in the opposition-in-exile, the Syrian National Council, pulled out of the coalition ahead of the talks saying it refused to negotiate with Assad.
Pitting Assad's regime, dominated by the Alawite offshoot of Shiite Islam, against largely Sunni Muslim rebels, the conflict has unsettled large parts of the Middle East.
Shiite Iran and its Lebanese militia ally Hezbollah have backed Assad; the mainly Sunni Arab Gulf states have supported the opposition; and the violence has often spilled over into neighbouring Lebanon and Iraq.
There were stark reminders of the conflict's impact in the run-up to the talks, with continued fighting on the ground and new evidence in a report alleging that Assad's forces have systematically killed and tortured 11,000 people.
One of its authors — the former chief prosecutor of the special court for Sierra Leone, Desmond de Silva — said it was the "smoking gun" proving "industrial-scale" killing by the Syrian regime.