Keith netted the only goal of a drab match in the 75th minute to end mid-table Chesterfield's unbeaten run of four matches.
Plymouth striker Keith swept home from close range following a corner by Kevin Watson to snatch the points for the Essex club.
Scotsman Keith had not scored in his first four matches for Colchester since his move from Plymouth at the beginning of the month, but has now scored two goals in two goals after netting the third from the penalty spot in the win at Peterborough on Good Friday.
If anything, it was Chesterfield who created the better of the scoring opportunities. In fact they should have broken the deadlock in the 20th minute when skipper Ian Evatt sliced a shot straight at home keeper Aidan Davison from point-blank range.
The visitors had an even better opportunity after just two minutes of the second half. Their two loan players combined with Leeds striker Jamie McMaster setting up Carlos Logan only for the Manchester City loanee to screw his shot over the bar.
Davison was the busier of the two keepers during the second half and he made one terrific block to deny McMaster with his legs.
Both managers made a couple of attacking substitutions in a bid to try and poach the victory but it was one of the surviving strikers still on the pitch, Keith, who came up with the winner.
Colchester now have 51 points and an excellent goal difference, so they have almost certainly secured their League One status for another season.
After a sticky period, Phil Parkinson's men have now taken 11 points from a possible 15.