Kitajima, who won the 100 and 200 breaststroke double at the 2004 and 2008 Olympics, rolled back the years to win the men's final in a Japanese record 58.90 seconds.
The 29-year-old squeezed out rival Ryo Tateishi by 0.70 of a second at Tokyo's Tatsumi Pool as both men booked their spots at this year's London Olympics.
"The Olympics only come once every four years - and that's as often as I break the record now," Kitajima, who flirted with retirement after Beijing, said. "Typical me!"
Kitajima was all business in the pool, however, and he smashed his fist into the water, letting out a roar of delight, after dominating Tateishi.
"The personal best will give me confidence going into the Olympics," said Kitajima, the former world record holder in the 100 and 200 metres.
"Now I have to work on turning it into the quickest time in the world."
Australia's Brenton Rickard currently possesses the world record of 58.58, set almost a year after Kitajima swam 58.91 at the 2008 Beijing Games.
"Eight years ago in Athens I had a rival I wanted to beat badly (American Brendan Hansen)," Kitajima said.
"Four years ago, I knew I would go to Beijing and be the world's best. This time was different. I knew I had to leave it all out there or else I wouldn't make it.
"I did that so it's a special feeling. People say I'm expected to waltz into the Olympic team but I put myself through some punishing training.
"I'll keep pushing to get closer to the world record and take on the world's best at the Olympics," added Kitajima, who races in the 200 later this week. "I don't like losing to anyone."
The Japanese nationals double as the country's Olympic qualifiers.