Abstract Sphingosine kinase2 (SphK2) is a type of sphingosine kinase which express highly in most of cancers. SphK2 produce sphingosine-1-phosphate (S1P) and then accumulate in cancer cells. Our previous study showed that S1P antagonized the effects of all-trans retinoic acid (ATRA) via the receptor-dependent and independent pathway. In this study, we aimed to investigate the roles of SphK2 in affecting ATRA's activity in human colon cancer cells. Cell proliferation was estimated by the clonogenic assay. The distribution of cell cycle was analyzed by flow cytometry assay. The apoptotic cells were determined by Annexin V-FITC/PI staining method. Western blotting assay was performed to analyze the levels of the proteins related to apoptosis and cell cycle. The mRNA levels of SphK2 and RARβ were evaluated by real-time PCR assay. RNA interference assay was performed to evaluate SphK2 activity. S1P antagonized the effect of ATRA on HT-29 cell proliferation, the ATRA-induced RARβ expression, the arrest of cell cycle in G1-phase, and induction of apoptosis. Down-regulation of SphK2 resulted in the reverse actions on the S1P-induced antagonistic effects on ATRA. Western blotting analysis indicated that down-regulation of SphK2 might activate apoptotic proteins, regulation of p53/p21Waf1/Cip1 and EGFR and PI3K/AKT signaling pathways. In conclusion, down-regulation of SphK2 increased the effects of ATRA on colon cancer cells.