Genome-wide association studies identified genetic susceptibility variants mostly lie outside of protein-coding regions. It suggested variants located at transcriptional regulatory region should play an important role in cancer carcinogenesis including lung cancer. In the present study, we systematically investigated the associations between the variants in the binding sites of an extensive transcription factor CTCF and lung cancer risk in Chinese population. A two-stage case-control design was conducted to evaluate the variants located at the uniform CTCF ChIP-seq peaks in a Chinese population (2,331 vs 3,077; 1,115 vs 1,346). The ChIP-seq data for CTCF, specified on lung cancer cell line A549, were downloaded from ENCODE database. Imputation was performed to increase the genome coverage in the CTCF binding regions. Three variants in CTCF binding sites were found to associate with lung cancer risk in the first stage. Further replication revealed a novel single nucleotide polymorphism rs60507107 was significantly associated with increased risk of lung cancer in two stages (Additive model: OR = 1.19, 95%CI = 1.11-1.27, P = 6.98 × 10(-7)). Our results indicate that rs60507107 in the binding site of CTCF is associated with an increased risk of lung cancer. This may further advance our understanding of regulatory DNA sequences in cancer development.