Two goals in the space of five first-half minutes from Phil Ifil and Anthony Wordsworth sank Brightin 2-1 and took Colchester up to third.
Midfielder Ifil broke the deadlock after nine minutes with a fierce shot from 25 yards before turning provider by crossing for Wordworth to head in from six yards.
Striker Kevin Lisbie put an overhead kick against the crossbar for Colchester before midfielder Gary Dicker reduced the deficit two minutes before the break by heading in a deflected cross from Nicky Forster.
Brighton pushed hard for an equaliser in the second half but impressive keeper Ben Williams pulled off a string of important saves to give Colchester a hard-earned victory.
Striker Glenn Murray should have hit the target for Brighton in the 76th minute when his left-foot shot was turned onto the post by Williams.
Williams also pulled off saves to deny Elliott Bennett and Forster.
A third successive home league defeat leaves Gus Poyet's Seagulls in the relegation zone.
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