SPHK1 overexpression increased invasive potential of esophageal carcinoma cells. We assessed the impact of gene transfection-mediated SPHK1 upregulation in EC9706 cells. (A) The two stable expression clones SPHK1-C17 and SPHK1-C24 both had markedly upregulated SPHK1 expression in EC970 cells. (B) In the transwell invasion assay, SPHK1 overexpression significantly increased the invasion of EC9706 cells. The numbers of invaded cells in the SPHK1 overexpressing cells (SPHK1-C17 group = 162.0 ± 16.52; SPHK1-C24 group = 173.0 ± 14.73) were higher compared with that of the vector group (37.33 ± 15.57), EC-9706 group (36.33 ± 4.933) and SPHK1-C7 group (31.33 ± 3.786); ANOVA P < 0.0001. (C) In 3D Matrigel culture assays, SPHK1 overexpression significantly increased the diameter and the invasive morphology of EC9706 cell clones. (D, F) SPHK1 overexpression significantly increased the proliferation of EC9706 cells. The OD450 values of the SPHK1-C17 group (3.664 ± 0.792) and SPHK1-C24 group (3.987 ± 0.632) were higher than that of the vector group (1.432 ± 0.453) and EC-9706 group (2.132 ± 0.334), ANOVA P < 0.031. (E, F) Annexin V assay showed overexpression of the SPHK1 gene did not influence the apoptosis level of EC9706 cells. The percentages of apoptotic cells in the SPHK1-C17 group (10.2 ± 0.2) and SPHK1-C24 group (7.3 ± 0.5) were comparable to that of the vector group (9.4 ± 3.2) and EC9706 group (7.3 ± 4.3), ANOVA P = 0.874.