Rab3D is a member of the ras-related GTP-binding protein Rab family and was found up-regulated in several types of cancer. However, little is known about the role of Rab3D in carcinogenesis and progression of esophageal squamous cell carcinoma (ESCC). Thus, in this study, we investigated the expression patterns and functional roles of Rab3D in human ESCC. We demonstrated that Rab3D was highly expressed in human ESCC cell lines. In addition, knockdown of Rab3D significantly inhibited the proliferation of ESCC cells and reduced the tumorigenesis in vivo. Moreover, knockdown of Rab3D significantly suppressed ESCC cell migration/invasion and accordingly alerted EMT related markers, which including up-regulated E-cadherin and down-regulated N-cadherin in ESCC cells. Finally, knockdown of Rab3D inhibited the levels of p-PI3K and p-Akt in ECA-109 cells. In conclusion, our data demonstrated that Rab3D functions as an oncogene in ESCC and knockdown of Rab3D suppressed ESCC cell proliferation and invasion, potentially through the PI3K/Akt signaling pathway. Overall, these findings suggest that targeting the Rab3D may be a potential therapeutic target for treatment of ESCC.