Purpose: Depth of wall invasion is an important prognostic factor in patients with gastric cancer, whereas the prognostic significance of intraoperative macroscopic serosal invasion (mSE) findings remain unclear when they show a discrepancy in pathologic findings. This study, therefore, assessed the prognostic significance of mSE. Methods: Data from cohort of 2,835 patients with resectable gastric cancer who underwent surgery between 1990 and 2010 were retrospectively reviewed. Results: The overall accuracy of mSE and pathologic results was 83.4%. The accuracy of mSE was 75.5% in pT2. On the other hand, the accuracy of pT3 dropped to 24.5%. According to mSE findings (+/-), the 5-year disease-specific survival (DSS) rate differed significantly in patients with pT2 (+; 74.2% vs. -; 92.0 %], pT3 (+; 76.7% vs. -; 72.8%) (P < 0.001 each), but not in patients with T1 tumor. Multivariate analysis showed that mSE findings (hazard ratio [HR], 2.275; 95% confidence interval [CI], 1.148-4.509), tumor depth (HR, 6.894; 95% CI, 2.325-20.437), nodal.status (FIR, 5.206; 95% CI, 2.298-11.791), distant metastasis (HR, 2.881; 95% CI, 1.388-6.209), radical resection (HR, 2.002; 95% CI, 1.017-3.940), and lymphatic invasion (HR, 2.713; 95% CI, 1.424-5.167) were independent predictors of 5-year DSS rate. Conclusion: We observed considerable discrepancies between macroscopic and pathologic diagnosis of serosal invasion. However, macroscopic diagnosis of serosal invasion was independently prognostic of 5-year DSS. It suggests that because the pathologic results could not be perfect and the local inflammatory change with mSE(+) could affect survival, a combination of mSE(+/-) and pathologic depth may be predictive of prognosis in patients with gastric cancer.