Objective? Pepsinogens have been previously studied as markers of gastric atrophy. The objective of this study was to investigate the clinical significance of the serum levels of pepsinogen (PG) I and II, as well as the pepsinogen I/II ratio (PGR) in the diagnosis of esophageal squamous cell carcinoma. Methods? A retrospective data analysis of patients who underwent gastroscopy and PG examination in Renmin Hospital was performed. The subjects were grouped into cancer and healthy control groups, and the differences in the serum levels of PGI and PGII, as well as the PGRs were compared. The receiver operating curve and the area under the curve (AUC) were also compared between the groups. Results? A total of 351 Chinese patients were enrolled in the study, 209 with esophageal squamous cell carcinoma and 142 healthy controls. Overall, the levels of PGI (P < 0.0001) and PGII (P = 0.0007), as well as the PGR (P = 0.007) of the cancer group were lower than those of the control group. Male subjects in the cancer group had lower PGI (P < 0.0001), PGII (P < 0.0001), and (P = 0.0138). The subjects < 65 years old in the cancer group showed lower PGI (P < 0.0001), PGII (P = 0.001), and PGR (P = 0.0087).Overall, these results show that the levels of PGI (AUC 0.64) and PGII (AUC 0.60) have a predictive ability for discriminating esophageal carcinoma. Moreover, in males < 65 years old, PGI (AUC 0.73) and PGII (AUC 0.69) also showed to have a predictive ability for discriminating esophageal carcinoma. Conclusion? Serum PG levels in patients with esophageal squamous cell carcinoma, especially in males aged < 65 years old, are lower than those in healthy people. PGI and PGII are useful for screening esophageal squamous cell carcinoma.