The relationship between vitamin B levels and the development and progression of lung cancer remains inconclusive. We aimed to investigate the relationship between B vitamins and intrapulmonary lymph nodes as well as localized pleural metastases in patients with non-small cell lung cancer (NSCLC). This was a retrospective study including patients who underwent lung surgery for suspected NSCLC at our institution from January 2016 to December 2018. Logistic regression models were used to evaluate the associations between serum B vitamin levels and intrapulmonary lymph node and/or localized pleural metastases. Stratified analysis was performed according to different clinical characteristics and tumor types. A total of 1498 patients were included in the analyses. Serum vitamin B6levels showed a positive association with intrapulmonary metastasis in a multivariate logistic regression (odds ratio (OR) of 1.016, 95% confidence interval (CI) of 1.002–1.031,p= 0.021). After multivariable adjustment, we found a high risk of intrapulmonary metastasis in patients with high serum vitamin B6levels (fourth quartile (Q4) vs. Q1, OR of 1.676, 95%CI of 1.092 to 2.574,p= 0.018,pfor trend of 0.030). Stratified analyses showed that the positive association between serum vitamin B6and lymph node metastasis appeared to be stronger in females, current smokers, current drinkers, and those with a family history of cancer, squamous cell carcinoma, a tumor of 1–3 cm in diameter, or a solitary tumor. Even though serum vitamin B6levels were associated with preoperative NSCLC upstaging, B6did not qualify as a useful biomarker due to weak association and wide confidence intervals. Thus, it would be appropriate to prospectively investigate the relationship between serum vitamin B6levels and lung cancer further.