Wilson, 57, took charge on Christmas Eve 2015, but after winning only six of their 26 league games this campaign the club are in the relegation zone.
Saturday's loss, their fourth in the last six games, left the Spireites in the drop zone on goal difference.
Left-back Ritchie Humphreys has been named as caretaker manager.
The 39-year-old, who is also the current chairman of the players' union, the Professional Footballers' Association, will be in charge for Tuesday's EFL Trophy game at Luton.
Former Sheffield Wednesday and Barnsley boss Wilson has managed in more than 1,000 competitive games and replaced Dean Saunders at the Spireites with the club 18th in League One.
Last term, Chesterfield finished in the same position they were in when Wilson took over, but are now 22nd in the table.
Following what proved to be his final game, Wilson said: "We're not playing badly. Yes, we're getting punished for opportunities we're giving to other teams but we're not playing badly.
"But it's about results, winning games, getting as many points as we possibly can and get us in a safe or safer position."
Director and company secretary Ashley Carson told BBC Radio Sheffield that Chesterfield did not want to be a club that had a "knee jerk reaction" when things were not going well.
But he said poor results, plus a dwindling fan base, had prompted the board's decision to relieve Wilson of his job.
"He had a great affinity for the club and he's an absolutely top drawer manager, but it gets to the stage where you look at everything that's happening and think 'we have got to change something'," Carson continued.
"We've just had to look at the bigger picture with all this, and feel the time is right, and that if we get a new (management) team in before the end of the month they can make some changes while the transfer window is open."
Chesterfield have sacked manager Danny Wilson and assistant Chris Morgan following Saturday's League One 2-0 defeat by Bradford City.