Background: The prevalence of gastric cancer is higher in males than in females. Reports have revealed that female gastric cancer patients exhibit some distinctive features, such as a high proportion of undifferentiated cancer and poor survival outcomes. The therapeutic effects of anti-PD-1 antibody have also been reported to be different between males and females. However, precise differences in molecular profiling features between males and females are yet to be reported. To elucidate sex differences in the clinicopathological and molecular biological characteristics of gastric cancer, we performed whole-exon sequencing and comprehensive gene expression analysis. Methods: We compared the clinicopathological factors and disease-specific survival (DSS) in 499 patients who underwent gastrectomy for gastric cancer between January 2014 and April 2019, excluding those with special types of gastric cancer and R2 resection. We also performed whole-exome sequencing using peripheral blood and tumor tissue, gene panel testing, and transcriptome analysis of tumor and nontumor tissues. Results: Among the 499 patients, 358 were male and 141 were female. Women had significantly more undifferentiated and M-region cancers (P < 0.05 for both). In terms of gene mutations, TP53 mutations were significantly more common in males (P = 0.002), whereas CDH1 (P = 0.005), PIK3CA (P = 0.046), and ERBB3 (P = 0.021) mutations were significantly more common in females. Expression profiles reflecting the tumor microenvironment were classified into two groups: T cell high group (290 patients) and T cell low group (209 patients), via cluster analysis. Men in the T-cell high group had significantly better DSS than those in the T-cell low group (P = 0.005), whereas no significant difference was observed among women in the T-cell high and low groups (P = 0.402). Among the 136 genes significantly upregulated in the T-cell high group, CCL18 (P = 0.048) gene was significantly upregulated in males and CD8A (P = 0.026) gene was upregulated in females. Conclusions: TP53 mutations were more common in male patients with gastric cancer, whereas CDH1, PIK3CA, and ERBB3 mutations were more common in female patients with gastric cancer. Additionally, women did not demonstrate good survival outcomes even in the T-cell high group considered to have enhanced tumor immunity.