The objectives of this work were to assess (i) the role of the SphK1/S1P signaling in various cellular models (U-2OS, SaOS-2, MG-63 and 143B) of osteosarcoma cell lines under hypoxia, (ii) the SphK1 enzymatic activity in human osteosarcoma samples versus nontumoral bones, and (iii) the expression of HIF-1α target gene GLUT-1, SphK1 and S1P1 in 130 cases of osteosarcomas (long and flat bone samples) as the link between SphK1/S1P signaling and hypoxia has never been investigated in human tissues. This evidence concerns the gene HIF1A and osteosarcoma.