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Purpose Rab27A and Rab27B, members of the Rab family of small GTPases, have aberrant expression and exert different roles in various cancers. However, their expression and potential prognostic values in esophageal squamous cell cancer (ESCC) still remain elusive. In the present study, we explored the association of Rab27A and Rab27B expression with clinical significance and prognosis in ESCC. Patients and methods A total of 100 surgically resected ESCC tissues were examined to evaluate Rab27A and Rab27B expression levels using the immunohistochemistry method. The relationship of Rab27A and Rab27B with clinicopathological features and prognosis was analyzed. We also investigated the correlation between Rab27A and Rab27B through external and internal validation. Results High-expression Rab27A was found to be significantly correlated with N (p=0.045) and TNM (p=0.005) stage, while up-regulated Rab27B was remarkably associated with N stage (p=0.033), TNM stage (p=0.009), and differentiation (p=0.013). High expression of both Rab27A and Rab27B had a worse overall survival (OS) rate. In addition, multivariate Cox regression analyses were utilized to validate that Rab27B expression is an independent prognostic factor for unfavorable OS. Further combined analyses showed that the Rab27Alow/Blow group had a superior OS rate than the Rab27Ahigh/Blow group, Rab27Alow/Bhigh group, and Rab27Ahigh/Bhigh group. Nevertheless, the latter three groups displayed rare significance between each two comparisons. Furthermore, our data demonstrated that Rab27A expression was positively correlated with Rab27B expression, which were also verified in TCGA datasets. Conclusion Rab27A and Rab27B expression levels could be potentially novel prognostic biomarkers in ESCC.