Dean Morgan's second-half penalty gave Chesterfield a hard-fought win to boost their survival chances while denting Sheffield Wednesday's promotion push.
John Bostock fouled Nicky Ajose and Morgan coolly dispatched the spot-kick.
Ryan Lowe, Reda Johnson and Lewis Buxton went close for the Owls in the first half, while Rob Jones had a header saved by Tommy Lee late on.
But Chesterfield had chances too, with Stephen Bywater denying Liam Ridehalgh and Drew Talbot.
It was Wednesday's third straight defeat in the league as they missed the chance to temporarily reclaim second place from city rivals Sheffield United.
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Chesterfield manager John Sheridan tells BBC Radio Sheffield:
"Other than the first 10 minutes, I thought Wednesday came out really strong and put us under a lot of pressure, which I expected, but I thought once we got in the game we really battled.
"I thought our second-half performance was really, really good and we came out looking to put them under a lot of pressure.
"Luckily for us we got the break with a penalty and I said to the lads stay in the game, keep it tight and that's what happened.
"We've shown great desire to keep a clean sheet near the end with Tommy [Smith] and the defenders defended really solidly.
"It's great for the fans, great to give us a boost beating one of the top teams and the main thing is it's three points."
Sheffield Wednesday manager Gary Megson tells BBC Radio Sheffield:
"I would say that is the best start to a game that we've had maybe since Tranmere, that was an exceptional start and today wasn't too far off, but against Tranmere we scored and today we didn't.
"It was decided by a soft penalty, not soft in terms that it was given, but soft in the way we defended it.
"The actual tackle itself was a little bit flimsy, shouldn't have been made and it's cost us the game.
"It's difficult to say an isolated incident has cost you the game - it probably has in terms of... we've obviously been beaten 1-0, but the amount of chances and the way we played in the first half was very good."
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