Background: Lung cancer is one of the most serious malignant tumors endangering human health and life. This study focused on evaluating the association between single nucleotide polymorphisms (SNPs) of the glutaminase (GLS) and lung cancer susceptibility in the Chinese Han population. Methods: A total of 684 lung cancer patients and 684 healthy individuals were enrolled. Five GLS SNPs (rs143584207 C/A, rs117985587 T/C, rs74271715 G/T, rs2355570 G/A, and rs6713444 A/G) were screened as candidate genetic loci. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to assess the association between GLS SNPs and lung cancer susceptibility. False-positive report probability (FPRP) analysis further verified whether the positive results deserved attention. Finally, the multi-factor dimensionality reduction (MDR) method was applied to analyze the interactions between SNPs. Results: The overall analysis revealed that GLS rs143584207 and rs6713444 were significantly associated with lung cancer susceptibility. The subgroup and clinical information analyses further revealed that GLS rs143584207 and rs6713444 could remarkably reduce lung cancer susceptibility in different subgroups (age >60, females, body mass index (BMI) 24, and lung adenocarcinoma). Rs143584207 could significantly reduce lung cancer susceptibility in non-smokers. Additionally, rs6713444 also had a protective effect on patients with advanced lung cancer. Conclusions: Our study indicated that GLS rs143584207 and rs6713444 could strikingly reduce lung cancer susceptibility in the Chinese Han population, which will give a new direction for the timely treatment of lung cancer.