Pannexin 1 (PANX1) is upregulated in many cancers, where its channel activity and signalling promote tumorigenic properties. Here, we report that potential internal translation start sites exist in mouse and human PANX1 which have implications in trafficking and protein interaction. Using mouse PANX1 constructs for each internal methionine (M) we saw that the shorter PANX1 isoforms were glycosylated, able to traffic to the cell surface and PANX1-M37 formed channels which could be activated by C-terminus cleavage or α1-adrenoceptor stimulation. Furthermore, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer, and glioblastoma multiforme. This isoform was increased upon