Flammulina velutipes has been reported to be beneficial in learning and memory capabilities, but the mechanisms underlying this remain unclear. In this study, Morris water maze and biochemical analyses of rat brain were used to evaluate the effects of F. velutipes polysaccharides (FVP) on scopolamine-induced learning and memory impairments. Results suggested that FVP significantly decreased the escape latency and total swimming distance of rats in the hidden platform test and increased the numbers of platform crossing and swimming distance of rats in the probe test. Biochemical examinations revealed that FVP significantly elevates SOD and GSH-Px activities, as well as neurotransmitter levels. The increased acetylcholine content owed to the increased acetylcholine acetyltransferase activity and decreased acetylcholinesterase activity. Moreover, learning and memory associated signalling pathways were activated by FVP elevating the expression of connexin 36 and p-CaMK II. These results conclusively proved that FVP is a potent agent against the progression of cognitive impairment.