Lee Sinnott becomes the second League Two manager to be fired this season following a 4-1 home defeat at the hands of Macclesfield Town.
Sinnott was appointed manager at Vale Park in November 2007 but failed to stop the Valiants from being relegated from League One.
Vale have since suffered a poor start to this season, losing three of their opening four home games.
His sacking comes just a week after ex Imps boss Alan Buckley lost his job at Grimsby Town.
Sinnott's assistant Dean Glover is set to be placed in temporary charge.