Objective: To investigate the effect of the calcium channel blocker verapamil on adipocyte differentiation and its mechanism of action. Methods: Preadipocytes from 3T3-L1 strain mouse embryos were cultured and differentiated into matured adipocytes in vitro. Verapamil was added to the culture medium in the concentration of 30 μmol/L on Day 0. Cell differentiation was determined by Oil Red O staining and marker gene mRNA expression was evaluated and compared by RT-PCR. The fluo-3/AM probe and laser scanning confocal microscopy were used to measure intracellular calcium concentrations. Results: ①The differentiation rate of 3T3-L1 preadipocytes exposed to verapamil was lower than that of untreated cells. ②Verapamil promoted the retention of pref-1 gene expression. Lipoprotein lipase expression in the verapamil group was significantly lower than that in the control group on Day 4, Day 6 and Day 8 (P < 0.05) and resistin expression was significantly lower than that in the control group on Day 6, Day 8 and Day 10 (P < 0.05). Fatty acid synthase expression in the verapamil group was significantly lower than that in the control group from Day 2 (P < 0.05). ③ Intracellular concentrations of calcium [Ca2+]i in the verapamil group were significantly decreased compared with those in the control group on Day 2, Day 4 and Day 6 (P < 0.05), while there was no obvious difference between the two groups on Day 0 (P > 0.05). Conclusion: In 3T3-L1 preadipocytes verapamil significantly reduced adipocyte differentiation, down-regulated the mRNA expression of three marker genes for adipocytes differentiation, and prolonged the mRNA expression of an inhibitor of differentiation. The inhibitory effect of verapamil on differentiation may involve its role as a blocker of calcium influx in adipocytes.