In an exclusive interview with ABC "World News" anchor Diane Sawyer, Ahmadinejad said there is no proof that the three Americans - Shane Bauer, Josh Fattal and Sarah Shourd – were not spies.
"How do you know they have accidentally crossed into Iran?" Ahmadinejad asked Sawyer. "How do you know they were looking for waterfalls and forests?"
It's been almost five months since the three were taken captive in Iran for allegedly straying across the country's border and almost two months since their families have had any word on their condition.
Their jailing comes amid an increasingly bitter standoff between the U.S. and Iran over Tehran's disputed nuclear program.
Ahmadinejad was combative in the interview and questioned Sawyer's facts. When Sawyer suggested that the evidence suggested the three hikers were simply tourists, the Iran president responded curtly: "Who has told you this? Are you a judge?"
Ahmadinejad interrupted when Sawyer tried to respond. "Just let me finish," he said. "Have the intelligence agents told you this?"
Iran's Foreign Minister said last week that the hikers will face trial in the Islamic republic on charges they were committing espionage.
Sarah Shourd, 31, along with Shane Bauer, 27, and Josh Fattal, 27 - all graduates of the University of California at Berkeley - had been trekking in Iraq's northern Kurdistan region when they accidentally crossed the border, according to their relatives.
"Sarah, Shane and Josh are good people," Sarah's mother Nora Shourd of Oakland, Calif., said in the video posted on a Web site dedicated to freeing the three. "They meant no harm to the Islamic Republic of Iran, and have a deep respect for your ancient and noble civilization. If they entered Iran, it was an innocent mistake."
The families previously have pleaded for their release to Ahmadinejad, but Friday's video was the first public outreach to Supreme Leader Ayatollah Ali Khamenei, who is Iran's highest-ranking political and religious authority.
"We're saying it's the holidays, please be compassionate and send them home," Shourd told The Associated Press.
On "The Early Show Saturday Edition," Nora Shourd told co-anchor Erica Hill, "We haven't gotten a response (from Iran to the video)yet, but we think it's a bit soon. The response has been mostly from this country so far. But we do hope that this kind of a plea will touch him and open up his heart enough to let our children come home for the holidays. It's really important to us."