Abstract: Temozolomide (TMZ) resistance remains a major challenge in the treatment of glioma, yet the underlying molecular mechanisms are incompletely understood. In this study, we identified calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) as a critical regulator of TMZ resistance in glioma. Analysis of clinical glioma specimens revealed CAMK2A expression was elevated in TMZ-recurrent patients. Functional experiments demonstrated that knockdown of CAMK2A markedly enhanced TMZ sensitivity, whereas overexpression of CAMK2A conferred resistance to TMZ in glioma cells. Mechanistically, CAMK2A phosphorylated Connexin 43 (CX43), thereby preventing its autophagic degradation and leading to CX43 stabilization. Notably, pharmacological intervention with Oroxylin A (OA) disrupted CAMK2A mediated CX43 phosphorylation by directly binding to CX43, resulting in enhanced TMZ sensitivity. Collectively, these findings uncover a previously unrecognized CAMK2A-CX43 signaling axis that drives TMZ resistance in glioma and highlight targeting CX43 phosphorylation as a potential therapeutic strategy to overcome chemoresistance.