BACKGROUND: Although the prognostic implications of negative lymph nodes (NLNs) has been reported for a variety of tumors, little information has been published about the NLNs in gallbladder cancer (GBC). PATIENTS AND METHODS: In this study, clinicopathological characteristics and survival times of patients who had undergone surgery for GBC were collected from the Surveillance, Epidemiology, and End Results Program-registered TNM stage database and analyzed. Univariate and multivariate Cox proportional hazards models were used to identify the predictors of survival. RESULTS: It was found that a cutoff of one to two NLNs is optimal when assessing the association with survival, survival rates being consistently better with two or more NLNs than with fewer than two. This optimal cutoff value of 2 was identified as an independent prognostic factor by univariate and multivariate analyses (all <0.001). Specifically, patients with two or more NLNs had better 5-year gallbladder cancer cause-specific survival than those with fewer than NLNs examined for stage I/II, stage III/IV, and all TNM stages (all <0.001). CONCLUSION: Our findings indicate that the number of NLNs is an independent prognostic factor after GBC surgery, and, together with the number of positive lymph nodes, this will provide better prognostic information than the number of positive lymph nodes alone.