The case, crucial for the under-fire court's future, opened at the Hague-based ICC with presiding Judge Chile Eboe-Osuji asking Ruto, 46 and fellow accused, Kenyan radio boss Joshua arap Sang, 38, to enter pleas to three charges of crimes against humanity each.
"Not guilty," both men told the judge.
Ruto came to court voluntarily from Nairobi to face charges of masterminding deadly post-election violence in Kenya, a bastion of stability in east Africa, after a tainted 2007 election.
Kenyan President Uhuru Kenyatta, a one-time political foe of Ruto's turned ally, goes on trial at the ICC on November 12. He also says he is innocent.
Ruto, dressed in a dark grey suit, gave a bemused smiled as the ICC's chief prosecutor Fatou Bensouda opened her case, while Sang, in a light grey suit, shook his head and occasionally smiled.
"Mr Ruto, as a powerful politician" planned the crimes "to satisfy his thirst for political power," Bensouda told the court.
"Mr William Ruto and Mr Joshua arap Sang are most responsible for these crimes," she said.
The ICC has come under increased pressure globally, especially from the 54-nation African Union, which accused the court of targeting the continent on the basis of race and demanded the court drop the Kenyan prosecutions.
The trial also comes just days after lawmakers in Kenya became the first in the world to approve moves to withdraw recognition of the 10-year-old court that so far has only one conviction under its belt.
Ruto and Sang each face three counts of murder, deportation and persecution after a wave of violence swept Kenya in 2007-08, leaving at least 1,100 dead and more than 600,000 homeless.
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