Background: Rs31489 in the cleft lip and palate transmembrane1-like gene (CLPTM1L) has been identified to be associated with lung cancer through genome-wide association studies (GWAS). However, some recent replication studies yielded inconclusive results. Thus, we undertook this study to investigate the precise effect of rs31489 on lung cancer susceptibility. Materials and Methods: A hospital-based case-control study in 1,673 Chinese subjects (611 individuals with lung cancer and 1,062 controls) and a meta-analysis among 32,199 subjects (16,364 cases and 15,835 controls) were performed in this study. Results: In our case-control study, rs31489 was inversely associated with lung cancer (AC versus CC: OR=0.68, 95% CI=0.52-0.88; additive model: OR=0.68, 95% CI=0.54-0.85; dominant model: OR=0.65, 95% CI=0.51-0.84). Stratification analysis by smoking status showed a significant association and strong genetic effect in non-smokers but not in smokers. Our meta-analysis further confirmed the association, although with significant heterogeneity contributed by study design and source of controls, as shown by stratified analysis. Sensitive and cumulative analyses both indicated robust stability of our results. In addition, there was no observable publication bias in our meta-analysis. Conclusions: Overall, the findings from our replication study and meta-analysis demonstrated that CLPTM1L gene rs31489 is significantly associated with lung cancer.