Chemotherapeutic agents are generally used as a frontline therapy for non-small cell lung cancer (NSCLC). However, resistance to chemotherapy arises rapidly in NSCLC, and the reasons for chemotherapy resistance have not been fully determined. Here, we found cisplatin, but not paclitaxel and doxorubicin, induced the enrichment of cancer stem cell (CSC) and conferred multidrug resistance in NSCLC cell lines. In vivo study confirmed drug-resistant tumors displayed the enhanced expressions of CSC transcription factors. Mechanistically, cisplatin treatment resulted in C/EBP-β-dependent increasing of TRIB1. The crucial role of TRIB1 in cisplatin-induced enrichment of CSC and drug resistance was verified by knockdown TRIB1. Interestingly, cisplatin treatment also contributed to the increasement of HDAC, the interaction of TRIB1 with HDAC, and inactivation of p53. Similarly, the silencing of HDAC led to reduction of cisplatin-induced CSC, and combined knockdown of HDAC and TRIB1 exhibited enhanced effect. Additionally, the combination of HDAC inhibitor and cisplatin showed a reinforced antitumor action in NSCLC cell lines with TRIB1-dependent manner and remarkably shrink tumors in xenograft models. Moreover, cisplatin-treated NSCLC patients with high levels of TRIB1 exhibited a significantly poorer prognosis. Our findings illustrate a novel perspective in the evolution of chemotherapy resistance and provide a promising approach for the treatment of patients with NSCLC.