Background: Currently, many detection methods have high sensitivity to the diagnosis of lung cancer. However, some postoperative patients with pulmonary nodules are eventually diagnosed as having benign nodules. The ideal evaluation of an individual with a pulmonary nodule would expedite therapy for a malignant nodule and minimize testing for those with a benign nodule. Methods: This case–control study is designed to explore the relationship between ACE1 rs4646994 polymorphism and the risk of lung cancer in patients with pulmonary nodules, for which 400 individuals with lung cancer and benign pulmonary nodules were included. A DNA extraction kit was used to extract DNA from peripheral blood. The relationship between ACE1 rs4646994 and the risk of lung cancer in patients with pulmonary nodules was determined by the chi-square test, logistic regression analysis and cross analysis. Results: The results showed that after adjusting for age and gender confounding factors, the risk of lung cancer in patients with pulmonary nodules carrying the DD genotype was more than three times that of the I carriers (II + ID) genotype (OR = 3.035, 95% CI, 1.252–7.356,p= 0.014). There was no significant difference between lung squamous cell carcinoma and lung adenocarcinoma in the polymorphism of ACE1 rs4646994 (p> 0.05). We also found that the ACE1 rs4646994 DD genotype frequency was inversely correlated with the risk of EGFR mutation in lung adenocarcinoma patients. Conclusions: Our study indicated that ACE1 rs4646994 polymorphism increases the risk of lung cancer in patients with pulmonary nodules from China.