Pregnane X receptor (PXR) is an important member of the nuclear receptor superfamily that copes with various endobiotic and xenobiotic stimuli, such as carcinogens by regulating an array of environmental response genes. Low PXR expression has been shown to promote tumor initiation and metastasis. The aim of the current study was to investigate whether the single nucleotide polymorphisms (SNPs) of PXR could alter lung cancer susceptibility in Chinese by affecting the function or expression of PXR. We genotyped three putatively functional SNPs of PXR (i.e., rs3814055C>T, rs3732360C>T, and rs3814058C>T) and analyzed their associations with lung cancer risk in a two-stage case-control study with a total of 1559 lung cancer cases and 1679 controls in the southern and eastern Chinese population. We found that in comparison to the rs3814058CC common genotype, the rs3814058T variants (TC/TT) which is located in the 3'-untranslated region (3'-UTR) of PXR conferred a consistently increased risk of lung cancer in both the southern Chinese (odd ratios (OR) = 1.24, 95% confidence interval (CI) = 1.03-1.49) and the eastern Chinese (OR = 1.33, 95% CI = 1.02-1.75). The variants also significantly interacted with smoking on increasing cancer risk (p = 0.023). Moreover, lung cancer tissues with the rs3814058T variants showed significantly lower PXR expression than those with rs3814058CC genotype in the smokers (p = 0.041). These results suggested that the rs3814058C>T polymorphism of PXR interacts with smoking on increasing lung cancer risk in Chinese smokers, which might be a functional genetic biomarker for lung cancer.