Sphingosine kinase 1 (SphK1) is an oncogenic enzyme promoting transformation, proliferation, and angiogenesis of a number of human tumors. However, its effect on hepatocellular carcinoma (HCC) behavior has not been fully clarified. The purpose of this study was to determine the correlation between HCC and SphK1, and to evaluate the effect of SphK1 inhibitor N,N-dimethylsphingosine (DMS) in HCC. The expression of SphK1 was measured in tissue samples from 76 HCC and paired adjacent noncancerous liver tissues (NT) by immunohistochemistry, quantitative real-time PCR, and Western blotting analysis. The effect of DMS was tested on HCC cells by evaluating cell viability in vitro. Transwell cell migration and invasion assay were carried out for functional analysis. Furthermore, Western blotting analysis was performed to examine the impact of DMS on the PI3K/Akt/NF-kB signaling. High expression of Sphk1 was observed in 84.21% (64/76) of the HCC versus 15.79% (12/76) of the adjacent non-tumorous liver tissues; the difference of Sphk1 expression between HCC and the adjacent non-tumorous liver tissues was statistically significant (P < 0.001). The results were confirmed by Western blot analyses and quantitative real-time PCR. DMS inhibited the proliferation of SK-Hep1 and MHCCLM3 cells which have a relatively high level of SphK1 in a time- and concentration-dependent manner, and the invasion and migration of SK-Hep1 cells were distinctly suppressed after undergoing treatment with DMS. Furthermore, DMS markedly suppressed the expression of phosphorylations of Akt and NF-κB in HCC cells. Our data suggest that the pathogenesis of human HCC maybe mediated by Sphk1, and the specific Sphk1 inhibitor DMS can play a therapeutic role in the treatment of HCC and thus, Sphk1 could represent selective targets for the molecularly targeted treatments of HCC.