Glucosamine (GlcN) is a natural amino monosaccharide in the human body, and evidence of its anticancer effects is growing. In this study, we aimed to evaluate the effects of GlcN for its cytotoxicity, MDR reversal effects and inhibitory effects on function and expression of P-glycoprotein (P-gp) transporter in the daunorubicin-resistant human gastric cancer cells. Cell viability was measured by MTT assay to evaluate the cytotoxicity and multidrug resistance (MDR) reversal effects of GlcN. The effects of GlcN on function and mRNA expression level of P-gp transporter were assessed by flow cytometry and real-time RT-qPCR, respectively. Our results indicated that GlcN reduced the proliferation of human gastric cancer cell line EPG85-257 and its drug-resistant variant EPG85-257RD in a dose-dependent manner. GlcN (at the concentrations of 0.5 and 1 mM) also enhanced the sensitivity of EPG85-257RDB cells to daunorubicin. The cellular accumulation studies showed that GlcN inhibited efflux activity of P-gp and enhanced the mean fluorescent intensity of Rho123 while ˙it had no effects on P-gp gene expression in these cells. This study suggested that the inhibition of P-gp activity is a novel mechanism of action by which GlcN could reverse MDR in EPG85-257RDB cells.