Abstract: Objective To investigate the mechanism and drug resistance of 26 S proteasome inhibitor bortezomib on multiple myeloma cell line RPMI8226 and myeloma side population(SP) cells. Methods The effect of bortezomib on the phosphorylation of PI3 K/AKT/mTOR signaling pathways was detected by Western blotting. The mechanism of resistance to bortezomib in RPMI8226 and SP cells was explored. Results The relative cell viability of SP cells at 48 h(3.62±0.28) was significantly higher than that of RPMI8226 cells(2.81±0.24, P=0.028). The clone formation ability results showed that SP cells were significantly stronger than RPMI8226 cells(P<0.05). Flow cytometry showed that bortezomib increased the S phase of both cells and decreased the G₂/M phase. The pro-apoptotic rate of RPMI8226(51.23±5.35) was significantly higher than that of SP cells(29.62±2.61, P=0.008). Western blotting results showed that bortezomib significantly reduced the phosphorylation levels of ART, mTOR and 4 E-RP1 in RPMI8226 and SP cells. The drug resistance results showed that SP cell resistance-related proteins P-gp, Caveolin-1(Cav-1), Fatty acid synthase(FASN) and CYP3 A4 expression were higher than those in RPMI8226 cells. Conclusion The inhibitory activity of bortezomib on RPMI8226 and SP cells is exhibited by cell apoptosis and phosphorylation levels of AKT and mTOR signaling proteins. The resistance is developed by the high expression of P-gp, CYP3 A4, Cav-1 and FASN.