To investigate the expression patterns of aquaporin 3 (AQP) 3 and AQP5, to evaluate their relationships with clinicopathological characteristics, and to determine their correlations with survival in esophageal squamous cell carcinoma (ESCC) patients, expression levels of AQP3 and AQP5 proteins in 126 ESCC patients were detected by immunohistochemistry. Then, paired Student's t-test, chi-square test, Spearman's rank correlation, Kaplan-Meier plots, and Cox proportional hazards regression model were used to analyze their associations with clinicopathological characteristics and survivals of ESCC patients. Both AQP3 and AQP5 proteins were localized on the cell membrane of tumor cells in ESCC tissues. The expression levels of two proteins in ESCC tissues were significantly higher than those in adjacent normal tissues (both P < 0.001). Additionally, high expression of AQP3 and AQP5 was both correlated with advanced invasion depth (both P = 0.01), aggressive lymph node status (P = 0.02 and 0.01, respectively), and positive distant metastasis (both P = 0.01). However, the overexpression of AQP3 and AQP5 alone did not influence the prognosis. Furthermore, ESCC patients with the co-expression of AQP3 and AQP5 showed the poorest prognosis in overall (P = 0.002) and disease-free survival (P < 0.001). Multivariate analysis showed that the co-expression of AQP3 and AQP5 was an independent prognostic factors of overall (P = 0.01) and disease-free survival (P = 0.01) in ESCC. Our data demonstrate for the first time that high expression of both AQP3 and AQP5, not each alone, is an independent poor prognostic factor in ESCC patients. Combined detection of the two proteins' expression may help to predict the progression and the prognosis of ESCC patients.