The purpose of this meta-analysis was to evaluate the relationship between serum 25-hydroxyvitamin D[25(OH)D] levels and gastric cancer. PubMed, Embase, Cochrane Library, Web of Science, The China Academic Journals full-text database, Wanfang Database of Chinese Academic Journals, VIP Chinese Science and Technology Periodicals database, and Chinese Biomedical Literature database were systematically searched. Case-control studies on the correlation between serum 25-hydroxyvitamin D levels and gastric cancer were retrieved, and the data extracted were analyzed. The results of 9 case-control studies containing 671 patients showed that serum 25(OH)D levels in the gastric cancer group were lower than those in the control group (weighted mean difference (WMD) = −8.90, 95% confidence interval (CI): −11.5, −6.32, p < 0.01); the risk of vitamin D deficiency in the gastric cancer group was higher than that in the control group (Odds ratio = 3.09, 95% CI: 1.96, 4.87, p < 0.01). The serum 25(OH)D levels in patients with well and moderately differentiated gastric cancer were higher than those in patients with poorly differentiated gastric cancer (WMD = −3.58, 95% CI: −6.41, −0.74, p = 0.01). Thus, low levels of vitamin D may increase the risk of gastric cancer. Systematic review registration: PROSPERO CRD42022327942.