Sphingosine-1-phosphate receptor 1 (S1PR1) is abnormally expressed in a variety of tumors. However, the clinical implications and biological roles of S1PR1 in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, we have focused on ESCC, and analyzed the expression of S1PR1 in human specimens at various histological grades of ESCC and the role of S1PR1 in Eca109 cells. Using human ESCC tissue microarray and immunohistochemistry, we found S1PR1 protein mainly located in the cytoplasm of cancer cells and normal esophageal mucosal epithelial cells, and small amounts in the plasma membrane. The levels of cytoplasmic S1PR1 in ESCC tissues were significantly higher than those in adjacent non-cancerous tissues. Cytoplasmic S1PR1 exhibited higher expression in ESCC tissues with poor differentiation than those with well differentiation. Conversely, the positive expression of plasma membrane S1PR1 was correlated with well differentiation. Kaplan-Meier survival analysis showed that patients with positive membrane S1PR1 expression tended to have longer survival time. Univariate and multivariate Cox regression analysis revealed that membrane S1PR1 expression was an independent prognostic factor for ESCC patients. Furthermore, overexpression of cytoplasmic S1PR1 promoted Eca109 cells from G1 phase to S phase and plasma membrane S1PR1 as the opposite, which may be associated with p21. Cytoplasmic S1PR1 signaling also promoted Eca109 cells migration. Our findings demonstrate that cytoplasmic S1PR1 plays an important role in the malignant behavior of human ESCC and may serve as a new target for ESCC therapy.