Platinum-based chemotherapy is the standard treatment for advanced non-small cell lung cancer (NSCLC); however, innate and acquired resistance is a major obstacle. To determine the transcriptional regulators of resistance, we first classified three-dimensional tumor spheroids derived from 11 NSCLC cell lines into cisplatin-sensitive or -resistant groups based on their cisplatin sensitivity and selected signature genes that were differentially altered between the groups. Using reverse engineering methods and functional validation, cAMP response element-binding protein 1 (CREB) was identified as a major regulator of cisplatin resistance. Among the putative target genes of CREB responsible for cisplatin resistance, cisplatin treatment significantly decreased the occupancy of CREB in the regulatory regions of TNKS and KDM6A in cisplatin-sensitive cells, but not in resistant cells, resulting in decreased expression of these protein in the sensitive group. Furthermore, CREB knockdown led to increased sensitivity to cisplatin with reduced levels of TNKS and KDM6A in both cisplatin-resistant tumor spheroids and tumors in a xenograft mouse model. In conclusion, our study delineates the role of CREB in cisplatin resistance and suggests that CREB inhibition is a potential therapeutic strategy for cisplatin-resistant NSCLCs.