Paclitaxel-based chemotherapy is widely used as the first-line treatment for non-small cell lung cancer (NSCLC). However, only 20%-40% of patients have shown sensitivity to paclitaxel. This study aimed to investigate whether P16 methylation could be used to predict paclitaxel chemosensitivity of NSCLC. Advanced NSCLC (N=45) were obtained from patients who were enrolled in a phase-III randomized paclitaxel-based clinical trial. Genomic DNA samples were extracted from the biopsies prior to chemotherapy. P16 methylation was detected using MethyLight. The association between P16 methylation and the sensitivity of paclitaxel in cell lines was determined by in vitro assay using a P16-specific DNA demethylase (P16-TET) and methyltransferase (P16-Dnmt). The total response rate of the low-dose paclitaxel-based chemo-radiotherapy was significantly lower in P16 methylation-positive NSCLCs than that in the P16 methylation-negative NSCLCs (2/15 vs. 16/30: adjusted OR=0.085; 95% CI, 0.012-0.579). Results revealed that P16 demethylation significantly decreased paclitaxel resistance of lung cancer H1299 cells (IC50 values decreased from 2.15 to 1.13 mu g/ml, P< 0.001). In contrast, P16-specific methylation by P16-Dnmt significantly increased paclitaxel resistance of lung cancer HCC827 cells and gastric cancer BGC823 cells (IC50 values increased from 18.2 to 24.0 ng/ml and 0.18 to 0.81 mu g/ml, respectively; P=0.049 and < 0.001, respectively). The present results suggest that P16 methylation may lead to paclitaxel resistance and be a predictor of paclitaxel chemosensitivity of NSCLC.