Insulin-like growth factor binding protein (IGFBP)-3 has both growth-inhibiting and growth-promoting effects at the cellular level. The cytotoxic action of several anticancer drugs is linked to increased ceramide generation through sphingomyelin hydrolysis or de novo biosynthesis. Herein, we investigated the role of IGFBP-3 on apoptosis of human umbilical vein endothelial cells (HUVEC) and its relationship with ceramide levels. We report that IGFBP-3 exerts dual effects on HUVEC, potentiating doxorubicin-induced apoptosis but enhancing survival in serum-starved conditions. Ceramide was increased by IGFBP-3 in the presence of doxorubicin and decreased when IGFBP-3 was added alone to cells cultured in serum-free medium. The protection exerted by the ceramide synthase inhibitor fumonisin B1 over doxorubicin-induced apoptosis was enhanced by IGFBP-3 with concomitant reduction of ceramide levels. IGFBP-3 alone activated sphingosine kinase (SK) and increased SK1 mRNA; the SK inhibitor N,N-dimethylsphingosine (DMS) blocked IGFBP-3 antiapoptotic effect. Moreover, IGFBP-3 increased IGF-I mRNA and dramatically enhanced IGF-I release. IGF-I receptor (IGF-IR) and its downstream signaling pathways Akt and ERK were phosphorylated by IGFBP-3, whereas inhibition of IGF-IR phosphorylation with tyrphostin AG1024 suppressed the antiapopoptic effect of IGFBP-3. Finally, IGFBP-3 increased endothelial cell motility in all experimental conditions. These findings provide evidence that IGFBP-3 differentially regulates endothelial cell apoptosis by involvement of the sphingolipid signaling pathways. Moreover, the survival effect of IGFBP-3 seems to be mediated by the IGF-IR.