Sam68 (Src associated in mitosis; 68 kDa) is an RNA-binding protein and substrate of Src family kinases. It is thought to play a role in cell cycle progression. Over-expression of Sam68 in fibroblasts was reported to have two separable functions dependent on its ability to bind RNA - cell cycle arrest or the induction of apoptosis. Post-translational modification with SUMO (small ubiquitin-like modifier) is common to many transcription factors and can regulate protein localization, stability and function. Here we show Sam68 to be modified by SUMO, and demonstrate that the SUMO E3 ligase (PIAS1) (protein inhibitor of activated STAT1) can enhance Sam68 sumoylation. Lysine 96, the first lysine in the amino-terminal region of Sam68, was found to be the major SUMO acceptor site. Mutation of the SUMO acceptor lysine to arginine enhanced the ability of Sam68 to induce apoptosis but inhibited its ability to act as a transcriptional inhibitor of cyclin D1 expression. A SUMO-1 Sam68 fusion protein, on the other hand, inhibited the ability of Sam68 to induce apoptosis but was a strong repressor of cyclin D1 expression. Thus, SUMO may be an important regulator of Sam68 function in cell cycle progression.