Glucosamine, a naturally occurring amino monosaccharide, has been reported to play a role in the regulation of apoptosis more than half century. However the effect of glucosamine on tumor cells and the involved molecular mechanisms have not been thoroughly investigated. Glucosamine enters the hexosamine biosynthetic pathway (HBP) downstream of the rate-limiting step catalyzed by the GFAT (glutamine:fluctose- 6-phosphate amidotransferase), providing UDP-GlcNAc substrates for O-linked ${eta}$-N-acetylglucosamine (O-GlcNAc) protein modification. Considering that O-GlcNAc modification of proteasome subunits inhibits its activity, we examined whether glucosamine induces growth inhibition $via$ affecting proteasomal activity. In the present study, we found glucosamine inhibited proteasomal activity and the proliferation of ALVA41 prostate cancer cells. The inhibition of proteasomal activity results in the accumulation of ubiquitinated proteins, followed by induction of apoptosis. In addition, we demonstrated that glucosamine downregulated proteasome activator $PA28{gamma}$ and overexpression of $PA28{gamma}$ rescued the proteasomal activity and growth inhibition mediated by glucosamine. We further demonstrated that inhibition of O-GlcNAc abrogated $PA28{gamma}$ suppression induced by glucosamine. These findings suggest that glucosamine may inhibit growth of ALVA41 cancer cells through downregulation of $PA28{gamma}$ and inhibition of proteasomal activity via O-GlcNAc modification.