Ruto and radio journalist Joshua arap Sang are accused of coordinating a campaign of bloodshed in the wake of Kenya's disputed 2007 election. Sang has also pleaded not guilty.
Kenyan President Uhuru Kenyatta is also due to face trial on similar charges in November, but is unlikely to appear beside Ruto in The Hague after claiming the East African country could not do without both leaders at the same time.
The unrest left around 1,100 dead and more than 600,000 displaced. The court's chief prosecutor Fatou Bensouda said Ruto engaged in crimes against humanity "to satisfy his thirst for political power."
"It is difficult to imagine the suffering or terror experienced by the men, women and children who were burnt alive, hacked to death or chased from their homes," Bensouda added.
"He [Ruto] gathered together an army of ... youths to go to war for him in the event the election was lost," Bensouda said.
"When the election was lost, he gave the order to attack."
Defense slams prosecution
But the ICC's claims have been rubbished by Ruto's lawyer Karim Khan, who labeled the charges "patently false."
"If the most basic investigations had taken place, the prosecutors would not have got us into this mess."
Ruto faced trial voluntarily after arriving from Nairobi on Monday. He was met at court by some 30 Kenyan MPs and other supporters, and his presence makes him the most senior serving politician to appear before the ICC.
The trial has caused controversy in Africa, with the Kenyan government passing a motion last week to withdraw its cooperation with the ICC. Supported by neighbors Uganda, the stance is part of an increasing sentiment on the continent that the ICC has a bias against African nations.
All 18 individuals indicted by the ICC since its establishment in 2002 hail from African countries.
ph/ccp (AFP, Reuters, dpa)