Second-half goals from David McGoldrick and Billy Sharp ensured the points went to Sheffield United at a bitterly cold Bramall Lane.
Wanderers looked in good shape after a solid first 45 minutes which saw them enjoy plenty of possession.
But decisive strikes from the Blades frontmen saw Wanderers taste defeat on their travels.
Phil Parkinson made two changes from the side that drew 1-1 with Reading at the University of Bolton Stadium on Tuesday evening. New signing Callum Connolly made his debut in place of Pawel Olkowski who missed out through injury, while Josh Vela returned to the starting XI at the expense of Craig Noone who was named among the substitutes.
Wanderers rode some early pressure from the hosts but soon got into their stride, particularly in midfield, where they started to get a foothold.
They won an early corner when O’Neil’s cross, intended for Donaldson at the near post, was cleared, but Connell’s set piece was over hit and it went out for a goal kick.
Matthews came off his line sharply to deny Bill Sharp from bearing in on goal after the Blades striker sprang into action after a ball from the left headed his way.
The hosts followed this up with three successive corners before the referee Keith Stroud blew up for a foul to give Wanderers some respite.
A block from Wheater in his own six-yard box prevented a powerful Stevens strike from testing Matthews in the Wanderers goal.
The game was played largely in the middle of the park with Wanderers enjoying plenty of possession and looked to break up play as quickly as possible when their opponents had the ball.
Wanderers were unfortunate not to have been awarded a corner when Connolly’s low long-distance shot appeared to deflect of a Blades’ defender’s boot.
David McGoldrick saw his powerful drive come off a Wanderers defender to earn his side a corner, but the visitors cleared their lines relatively easy.
United were ending the half strongly and Fleck saw a firmly hit effort canon off O’Neil for another Blades corner.
Wheater was on hand to break another attack from the hosts, this time diverting McGoldrick’s reverse pass behind for a corner.
Wanderers had a bright start to the second half and enjoyed two successive corners to keep the Blades on their toes.
Donaldson had a strong chance to put Wanderers in front when he leapt to get his head on Connell’s perfectly weighted pass, but he was unable to get enough power to steer the ball beyond Henderson.
The hosts found themselves in front on 56 minutes through McGoldrick who struck the ball into an empty net when Sharp burst into the box after shaking off the attention of Hobbs with Matthews already committed.
Some lovely play on the right hand side involving Lowe saw Vela charge into the box and laying on the ball for Ameobi but he was crowded out before he could guide his shot on goal.
Parkinson made his first change with twenty minutes remaining when he brought on Magennis for Vela.
Connolly had a good chance to net an equaliser after being played in by Taylor but the on-loan midfielder placed his shot over Henderson’s bar.
Sharp doubled the Blades lead after 73 minutes when he was the first to get to Norwood’s excellent free-kick. The striker powered in a header from close range leaving Matthews with no chance.
Bolton Wanderers: Matthews, Taylor, Lowe, Vela (Magennis 69), Donaldson, Ameobi, Hobbs, Connolly, O’Neill, Wheater (C), Connell
Subs Not Used: Buckley, Noone, Murphy, Wilson, B. Williams, Grounds.