Rare germline variations contribute to the missing heritability of human complex diseases including cancers. Given their very low frequency, discovering and testing disease-causing rare germline variations remains challenging. The tag-single nucleotide polymorphism rs17728461 in 22q12.2 is highly associated with lung cancer risk. Here, we identified a functional rare germline variation rs548071605 (A>G) in a p65-responsive enhancer located within 22q12.2. The enhancer significantly promoted lung cancer cell proliferation in vitro and in a xenograft mouse model by upregulating the leukemia inhibitory factor (LIF) gene via the formation of a chromatin loop. Differential expression of LIF and its significant correlation with first progression survival time of patients further supported the lung cancer-driving effects of the 22q-Enh enhancer. Importantly, the rare variation was harbored in the p65 binding sequence and dramatically increased the enhancer activity by increasing responsiveness of the enhancer to p65 and B-cell lymphoma 3 protein, an oncoprotein that assisted the p65 binding. Our study revealed a regulatory rare germline variation with a potential lung cancer-driving role in the 22q12.2 risk region, providing intriguing clues for investigating the "missing heritability" of cancers, and also offered a useful experimental model for identifying causal rare variations.© 2021 Wiley Periodicals LLC.