The 17-year-old schoolgirl clocked 3:58.37 seconds to beat her previous mark by 0.49sec.
Ledecky only broke the previous world record in the event at the US Championships on August 9 and continues her remarkable run of world records.
She holds the world records in the 400m, 800m and 1500m freestyle to make her the first woman since Janet Evans to hold all three records at the same time.
Ledecky's win in the 400m was her fourth gold medal of the Pan Pacific Championships on the Gold Coast after wins in the 200m and 800m freestyle and as part of the USA 4x200m freestyle relay.
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