Objective To investigate whether etomidate (ETO) affects the malignant biological behaviors of esophageal cancer (EC) cells by regulating the sphingosine kinase 1/sphingosine-1-phosphate/sphingosine-1-phosphate receptor 3 (SPHK1/S1P/S1PR3) signaling pathway. Methods EC cells KYSE30 were divided into NC group, ETO group, pc-NC group, pc-SPHK1 group, ETO+pc-NC group, and ETO+pc-SPHK1 group. The SPHK1 mRNA expression, proliferation, apoptosis, migration, invasion, CXCL-2 and CXCL-8 levels, B7H1, E-cadherin, N-cadherin, vimentin, PCNA, cleaved caspase-3, SPHK1, S1P, S1PR3 proteins in KYSE30 cells were detected. A nude mouse transplant tumor model was established, and the effect of ETO on transplant tumor growth was observed. Results Compared with the NC group, the ETO group exhibited significantly decreased mRNA expression of SPHK1, OD490 values at 24 h and 48 h, numbers of migratory and invasive cells, levels of CXCL-2 and CXCL-8, and protein expressions of B7H1, N-cadherin, vimentin, PCNA, SPHK1, S1P and S1PR3 in KYSE30 cells, while significantly increased apoptosis rate and protein expressions of E-cadherin and cleaved caspase-3 (P<0.05). Compared with the pc-NC group, the pc-SPHK1 group showed increased SPHK1 mRNA, OD 490(24h, 48h) values, cell migration and invasion numbers, CXCL-2 and CXCL-8 levels, B7H1, N-cadherin, vimentin, PCNA, SPHK1, S1P, and S1PR3 proteins in KYSE30 cells, while decreased apoptosis rate, E-cadherin and cleaved caspase-3 〖JP2〗proteins (P<0.05). Overexpression of SPHK1 was able to weaken the inhibitory effect of ETO on the malignant biological behaviors of KYSE30 cells (P<0.05). Nude mouse xenograft experiments confirmed that ETO inhibited tumor growth and promoted apoptosis of tumor tissue cells(P<0.05). Conclusion ETO can inhibit the malignant biological behaviors of KYSE30 cells, which may be related to the inhibition of the SPHK1/S1P/S1PR3 signaling pathway