The 33-year-old, who captured double gold in the men's 100 and 200 metres breaststroke at the 2004 and 2008 Olympics, flexed his muscles by qualifying fastest for Tuesday's 100m final with a time of 59.62 seconds at Tokyo's Tatsumi Pool.
Japan's latest swimming sensation Kosuke Hagino, meanwhile, booked his place at this summer's Rio Olympics with an equally convincing victory in the men's 400 metres medley.
A chiselled-looking Kitajima became the first Japanese swimmer to qualify for four Olympics four years ago and demonstrated he was not done yet with another strong swim in his semifinal heat.
"I wanted a slightly quicker time," said Kitajima, who nonetheless landed a psychological blow by edging out young pretender Yasuhiro Koseki (1:00.09) and narrowly dipping under the Olympic qualifying time of 59.63.
"But it's been a while since I swam 59-something. I feel this is a reward for all the hard work I've put in. I'd still like to set a personal best again in the final."
