In this study, we have investigated the role of a glioma-specific cation channel assembled from subunits of the Deg/epithelial sodium channel (ENaC) superfamily, in the regulation of migration and cell cycle progression in glioma cells. Channel inhibition by psalmotoxin-1 (PcTX-1) significantly inhibited migration and proliferation of D54-MG glioma cells. Both PcTX-1 and benzamil, an amiloride analog, caused cell cycle arrest of D54-MG cells inG 0/G 1 phases (by 30 and 40%, respectively) and reduced cell accumulation in S and G 2/M phases after 24 h of incubation. Both PcTX-1 and benzamil up-regulated expression of cyclin-dependent kinase inhibitor proteins p21 Cip1 and p27 Kip1. Similar results were obtained in U87MG and primary glioblastoma multiforme cells maintained in primary culture and following knockdown of one of the component subunits, ASIC1. In contrast, knocking down δENaC, which is not a component of the glioma cation channel complex, had no effect on cyclin-dependent kinase inhibitor expression. Phosphorylation of ERK1/2 was also inhibited by PcTX-1, benzamil, and knockdown of ASIC1 but not δENaCinD54MGcells. Our data suggest that a specific cation conductance composed of acid-sensing ion channels and ENaC subunits regulates migration and cell cycle progression in gliomas. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.