Colchester capitalised on Portsmouth's early defensive frailties to earn their first away league win since October.
The U's were ahead after just two minutes when Gavin Massey turned home Freddie Sears's cross, but Jed Wallace volleyed home an equaliser soon after.
Pompey debutant Sam Sodje then felled Sears in the box, who scored the penalty himself before Massey struck the visitors' third after 20 minutes.
James Keene blasted home to reduce the deficit, but the U's held on.
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Portsmouth caretaker manager Guy Whittingham told BBC Radio Solent:
"There was no commitment to anything, you can't excuse that in a football game.
"It's what football is about, it's what makes winners and losers and we had none of that in the first half.
"We try to tell them (the players) that the fans will support you, all you've got to do is be committed.
"They'll give and take on the losing front but if you don't commit that's when they're not happy."
Portsmouth 13 Eastwood 04 Sodje 10 Moutaouakil 18 Ertl 33 Rocha (Gyepes - 27' ) 34 Butler 03 Cooper (Reed - 68' ) 35 Wallace 09 Connolly (Walker - 46' ) 17 Keene 21 Harris Substitutes 01 Smith 16 Gyepes 22 Webster 06 Walker 07 Reed 14 Sutherland Colchester United 44 Walker 02 Wright 04 Okuonghae 20 Wilson 25 White 40 Thompson 41 Clifford (Bond - 86' ) 39 Porter Booked (Wright - 82' ) 11 Sears (Bean - 72' Booked ) 17 Ibehre Booked 21 Massey Substitutes 01 Cousins 18 Eastman 14 Bond 15 Bean 30 Wright 09 Morrison 33 Compton