The objective of this study was to investigate the effect of the new generation of tyrosine kinase inhibitor flumatinib mesylate on C-MYC, HIF-1α and VEGF in multiple myeloma (MM) cell line U266. Different concentrations (1, 5, 10 µmol/L) of flumatinib mesylate were used to act on U266 cell line for 8, 16 and 24 h, and the expression of C-MYC, and HIF-1α genes was detected by real-time fluorescence-quantitative PCR, the expression of C-MYC, HIF-1α and VEGF was measured by Western blot. The results showed that the gene expression of C-MYC and HIF-1 genes decreased gradually with the increasing of flumatinib mesylate concentration (P < 0.05). At the same concentration of flumatinib mesylate, the expression of C-MYC and HIF-1α gene decreased gradually with prolonging of treatment time with the flumatinib mesylate (P < 0.05). When the flumatinib mesylate acted the U266 cell line for 16 h, the expression of C-MYC, HIF-1α and VEGF decreased gradually with the increasing of flumatinib mesylate concentration (P < 0.05). It is concluded that the flumatinib mesylate can reduce the expression of C-MYC, HIF-1 α and VEGF in U266 cell line in a time- and dose-dependent manners, so flumatinib mesylate may become a new drug for MM therapy.