The triple gold medallist from both the 2008 Beijing and 2012 London Olympics easily won his heat in 46.71 seconds at the Camperdown Classic.
But he had to settle for third place overall after his team-mates Warren Weir and Yohan Blake both posted faster times in their heats.
Weir, who finished third behind Bolt and Blake in the 200m final at London last year, won in a personal best time of 46.21 seconds.
Blake, racing in the same heat as Weir, stopped the clock at 46.64 after fading in the final straight following a blistering start.
There was no final for the event, which was part of a meeting run by the Racers Track Club that attracted around 30 athletes.
Bolt is the world record holder and double Olympic champion for the 100 and 200 sprints but often competes in 400 races at the start of each season to build his fitness.
Next week, he is scheduled to appear in Texas for the National Basketball Association All-Star Game but his major goal this year is the world championships in Moscow.
- Sports & Recreation
- Athletics, Track & Field
- Yohan Blake
- 2012 London Olympics