Background: Multiple myeloma is featured by the proliferation of malignant plasma cell in bone marrow. We aimed to demonstrate the effects of valproic acid combined with GANT61 on multiple myeloma cell proliferation and clarify its mechanism. Material/Methods: Multiple myeloma cells were exposed to valproic acid, GANT61, or the combination of valproic acid and GANT61, respectively. MTT assay was performed to detect the cell viability. Quantitative reverse transcriptase polymerase chain reaction and western blotting were used to detect mRNA and expression levels of proteins in Hedgehog signaling pathway. The Q-value of the combination regime was calculated to evaluate the drug combination effect. Results: Both valproic acid and GANT61 alone inhibited multiple myeloma cell proliferation in a dose-dependent manner compared to the control. In the presence of GANT61 or not, valproic acid inhibited multiple myeloma cell proliferation in a time-dependent manner. These 2 drugs had a synergistic effect at valproic acid concentration of ³4 mM. Expression analysis showed that valproic acid significantly inhibited the expression levels of PTCH1, GLI1, and HES-1. GANT61 enhanced the inhibition of Hedgehog signaling pathway mediated by valproic acid. Conclusions: GANT61 and valproic acid inhibited multiple myeloma cell proliferation synergistically by inhibiting the Hedgehog signaling pathway. The present study may provide a combination regime for the therapy of multiple myeloma.