Former Ipswich Town defender Brown came up trumps in the 62nd minute to end the challenge of 10 man Tranmere, and so boost his side's hopes of filling the second spot behind runaway leaders Southend.
Tranmere, who are not out of the woods, at the other end of the table, face a big mountain to climb when their midfielder Stephen O'Leary was sent off after only 25 minutes.
O'Leary ploughed into Colchester full back John White with a late, two-footed challenge and was duly shown the red card for serious foul play.
Rovers therefore faced the prospect of playing the final 65 minutes with just 10 men, and their resistance was finally broken when experienced substitute Jason McAteer brought down star man Mark Yeates just outside the penalty area.
Brown had failed to seriously test Steve Wilson with an earlier free kick, but this time his 20 yard effort soared over the defensive wall and into the roof of the net, giving Rovers keeper no chance.
It was only Brown's second League goal of the season following his strike in the 5-0 thumping of Gillingham in late November. He will rarely have scored a more important goal in his long career than the one that lifted the U's to second spot.
Tranmere rallied during the closing stages and they had a couple of chances to steal an unlikely equaliser.
Impressive midfielder Steve Jennings was agonisingly close to diverting home a cross from David Raven, and near the end he headed inches wide from a similar cross by McAteer.
Leading scorer Chris Greenacre had an opportunity in stoppage time, but he lost control of the ball at the final moment and Liam Chilvers hacked to safety.
That was Rovers last chance, while at the other end substitute Tony Thorpe blasted over the bar with just keeper Wilson to beat.
Blackpool's late equaliser at Brentford means that Colchester are in second spot, at least for 24 hours. Huddersfield, another of their promotion rivals, entertain Chesterfield on Tuesday evening and if they can win by at least two goals then they will claim second spot.
It is proving to be a thrilling promotion scramble and the U's will look to continue their winning trend at Bournemouth this weekend.