MicroRNA-26a (miR-26a) has been reported to be involved in the tumorigenesis of several tumors, but its biological function and molecular mechanism in multiple myeloma (MM) are still unknown. In this study, we found that overexpression of miR-26a obviously inhibited MM cell growth, and delayed tumor growth in xenografts. Further studies showed that overexpression of miR-26a induced cell cycle arrest at G0/G1 phase in MM cells. MiR-26a mimic down-regulated the expression levels of CDK6 and E2F1, but up-regulated p53 and p21 expression. In contrast, overexpression of CDK6 decreased the effect of miR-26a mimic on MM cell survival. Moreover, miR-26a targeted CDK6 mRNA and thus suppressed CDK6 protein expression. Overexpression of miR-26a also enhanced the cytotoxic action of doxorubicin against MM. These results demonstrated that miR-26a was involved in the development of MM through regulating CDK6 signaling pathway, and indicated that miR-26a could be as a novel target for anti-tumor therapy in clinic as a single strategy or in combination with other anti-tumor drugs in MM.