The 86-year-old ex-general said that prosecutors haven't proven his participation in the killings.
"I declared myself innocent," Rios Montt told the three-judge tribunal as many in the audience applauded. "It was never my intention or my goal to destroy a whole ethnic group."
"I never ordered attacks on a specific race, I never did anything I'm being accused of and my participation in that has not been proven," he added.
Rios Montt is being tried together with Jose Rodriguez Sanchez, a 68-year-old former general who was a high-ranking member of the military chiefs of staff during Rios Montt's administration.
On Wednesday, prosecutors asked that both men be sentenced to 75 years in prison.
Francisco Garcia Gudiel, Rios Montt's defense lawyer, denied the charges during closing arguments Thursday.
"My defendant never ordered, never ran, never planned, and never oversaw what he is accused of doing," he said.
Rios Montt seized power in a March 23, 1982, coup, and ruled until he was overthrown just over a year later.
Prosecutors say that while in power, Rios Montt was aware of, and thus responsible for, the slaughter of at least 1,771 Ixil Mayas in the towns of San Juan Cotzal, San Gaspar Chajul and Santa Maria Nebaj in Guatemala's western highlands.