Resistance to chemotherapy remains a critical barrier to effective cancer treatment. Although cisplatin is one of the most commonly used chemotherapeutic agents in the treatment of non-small cell lung cancer (NSCLC), mechanisms of resistance to this drug are not fully understood. Here, we report a novel cisplatin-resistance mechanism involving SET Domain Containing 2 (SETD2), a histone H3 lysine 36 (H3K36) trimethyltransferase, and cAMP-responsive element-binding protein 1 (CREB1). A549 cells selected in vivo to give brain metastases exhibited cisplatin resistance and decreased expression of phosphorylated CREB1. Next-generation sequencing (NGS) analysis identified a missense mutation in SETD2 (p.T1171K), and we demonstrated that SETD2-mediated trimethylation of H3K36 (H3K36me3) and CREB1 phosphorylation are critical for cellular sensitivity to cisplatin. Moreover, we showed that suppression of SETD2 or CREB1 and ectopic expression of mutant SETD2 conferred cisplatin resistance through inhibition of H3K36me3 and ERK activation in NSCLC cells. Our results provide evidence that SETD2 and CREB1 contribute to cisplatin cytotoxicity via regulation of the ERK signaling pathway, and their inactivation may lead to cisplatin resistance.