Glioblastoma (GBM) is the most lethal malignant primary brain tumor. Although multimodal therapy has been applied for GBM, the median survival time remains less than 16 months. Thus, better therapeutic targets in GBM are urgently needed. Herein, we first identified five new N-terminal-truncated Cx32 isoforms (GJB1–28k, GJB1–22k, GJB1–20k, GJB1–15k, and GJB1–13k) and further demonstrated that they were generated via cap-independent internal translation through internal ribosome entry sites (IRESs) in the coding sequence of GJB1 mRNA. Among these isoforms, GJB1–13k inhibited proliferation, promoted apoptosis, and limited cell cycle progression in GBM cells by inhibiting global mRNA translation. In vivo experiments further confirmed the antitumor activity of GJB1–13k against GBM cells. In addition, TSR3, a ribosomal maturation factor, was demonstrated to directly interact with GJB1–13k. Moreover, GBM cells with high TSR3 expression exhibited low sensitivity to GJB1–13k treatment, while GJB1–13k sensitivity was restored by TSR3 knockdown. Our work identifies a new IRES-mediated protein, GJB1–13k, and suggests that overexpression of GJB1–13k in GBM cells with low TSR3 expression or combined targeting of GJB1–13k and TSR3 in GBM cells with high TSR3 expression constitutes a potential therapeutic strategy for GBM.