A chiseled-looking Kitajima, who won the 100 and 200 breaststroke double at the 2004 and 2008 Olympics, clocked the fastest time of 59.31 seconds at Tokyo's Tatsumi Pool.
"I felt really smooth this morning in the heats and I pretty much picked up where I left off," Kitajima explained at the nationals, which double as Japan's Olympic qualifiers.
"It doesn't matter how tomorrow's final turns out, this gives me a lot of confidence. I wanted to be at the turn in 27 seconds and not run out of steam at the end and I did it."
The 29-year-old added: "The 100 is fun. Some people say I have a better chance in the 200 but I'm not ready to give up on the 100 yet."
Rival Ryo Tateishi set a time of 1:00.04 in the first semi-final.
"It will be real fight against Kosuke tomorrow," said Tateishi. "But it's what I've been working towards."
Four swimmers booked their places for this year's London Olympics on day one, Kosuke Hagino and Yuya Horihata in the men's 400 individual medley and Miyu Otsuka and Miho Takahashi in the women's 400 IM.
The 17-year-old Hagino set a Japanese record of 4:10.26 - 1.78 seconds under Horihata's old mark - to become the first high school student to make the men's team since Kitajima and Jiro Miki qualified for the 2000 Sydney Olympics.