Accumulating evidence has demonstrated that voltage-gated potassium channels (Kv channels) were associated with regulating cell proliferation and apoptosis in tumor cells. Our previous study proved that the Kv channel blocker 4-aminopyridine (4-AP) could inhibit cell proliferation and induce apoptosis in glioma. However, the precise mechanisms were not clear yet. MicroRNAs (miRNAs) are small noncoding RNAs that act as key mediators in the progression of tumor, so the aim of this study was to investigate the role of miRNAs in the apoptosis-promoting effect of 4-AP in glioma cells. Using a microRNA array, we found that 4-AP altered the miRNA expression in glioma cells, and the down-regulation of miR-10b-5p induced by 4-AP was verified by real-time PCR. Transfection of miR-10b-5p mimic significantly inhibited 4-AP-induced caspases activation and apoptosis. Moreover, we verified that apoptosis-related molecule Apaf-1 was the direct target of miR-10b-5p. Furthermore, miR-10b-5p mimic significantly inhibited 4-AP-induced up-regulation of Apaf-1 and its downstream apoptosis-related proteins, such as cleaved caspase-3. In conclusion, Kv channel blocker 4-AP may exert its anti-tumor effect by down-regulating the expression of miR-10b-5p and then raised expression of Apaf-1 and its downstream apoptosis-related proteins. Current data provide evidence that miRNAs play important roles in Kv channels-mediated cell proliferation and apoptosis.