Dave Jones maintained his unbeaten start at the S6 helm but had to settle for a point as Colchester frustrated the promotion-chasing Owls at the Weston Homes Community Stadium.
The U's took an early lead through Ian Henderson and although Miguel Llera claimed his third goal in as many games, Wednesday emerged from Essex having lost ground on rivals Sheffield United for the second automatic slot.
Wednesday went into the contest on a stunning run of form that yielded seven wins from the previous eight games but got off to the worst possible start.
Barely five minutes in, Henderson ran into space inside the penalty area to take a pass from Steven Gillespie into his stride and flick a low shot past Stephen Bywater.
The goal was Wednesday's first concession in five and as the early exchanges unfolded, a scrappy contest developed with the visitors posing less of a threat in the final third as has been the case of late.
But that changed on the 20-minute mark when the Owls created two chances inside a minute.
First, Chris Lines sent a dangerous free kick into the area where Gary Madine flicked a header wide, then Nile Ranger failed to get a proper connection six yards out following good work down the right by Michail Antonio.
Lines drilled a low shot into the arms of Ben Williams two minutes later as the Owls ramped up the pressure but Colchester weathered the mini storm to edge back into proceedings.
But opportunities at either end were few and as half time approached, it seemed clear that both sides had more to give in the second period.
And Wednesday demonstrated exactly that six minutes in when Llera struck from close range to level the score.
Madine did exceptionally well to win a free kick deep down the left flank and Lines delivered an accurate feed right into the heart of the danger zone, where Llera was on hand to stab into the back of the net.
The Owls had their tails up and a Madine header brushed the top of the net moments after the restart before Jermaine Johnson saw a pacey shot deflected wide.
The lion's share of play was in the home side's half as Wednesday went in search of a second goal but the impetus from the equaliser subsided as the game went on.
Johnson and Lines had half chances during the closing stages but little would drop for the Owls, who left Essex frustrated and in need of a dramatic Sheffield swing to keep their top-two ambitions alive.
Colchester: Williams, Okuonghae, Gillespie, Izzet (Odejaye 74), Bond, Henderson (O'Toole 87), Eastman, Wilson, Wordsworth, White, Duguid
Unused substitutes: Rose, Heath, Sears
Owls: Bywater, Buxton, Llera, Batth, Beevers, Antonio, Lines, Semedo, J Johnson, Madine, Ranger (O'Grady 72)
Unused substitutes: Weaver, D Jones, Prutton, M Jones