The election, triggered by the death of the constituency's Labour member of parliament, is being closely watched as an indicator of Brown's diminishing appeal 11 months after he took over as prime minister from Tony Blair.
Brown's popularity ratings have collapsed since October after he backed away from calling an early election.
Some Labour members are asking whether he is the best man to lead them into a parliamentary election due by 2010.
The Conservatives believe victory in this Labour heartland would signal that the political wind has turned in their favor, putting them on track to unseat Brown.
More recently, Labour suffered a drubbing in May 1 local council elections and is struggling to restore public confidence after a botched tax reform left many of the poorest worse off.
Rising living costs and anxiety over a potential housing market slump have added to the government's woes.
Lifelong Labour voters on the streets of Crewe and the neighboring market town of Nantwich on Thursday blamed Labour for the rising cost of living and said they had had enough.
While the election was for a single parliamentary seat, voters were aware of its significance for the national picture.
"I think Labour had lost it before this," said taxi driver Terry Clorley, 63, referring to the next general poll. Clorley said he voted Conservative for the first time on Thursday.
"They must know the writing is on the wall," said 60-year-old Patrick Sutton, also a lifelong Labour supporter who switched to Conservative candidate Edward Simpson.
Brown could now face a renewed backlash from members of his Labour Party who already doubt his ability to win.
The single-seat election was triggered by the death of Labour incumbent Gwyneth Dunwoody, who held it for 34 years. Her daughter Tamsin, a 49-year-old single mother, ran for Labour.
Voters said the rising cost of food and fuel, the government's mishandling of tax reform and a negative campaign run by Labour had prompted the move to the Conservatives.
Analysts will now probe the numbers to see how big the swing was from Labour to the Conservatives and what that means for a future national election.