Sirtuin deacetylases and FOXO (Forkhead box, class O) transcription factors have important roles in many biological pathways, including cancer development. SIRT1 and SIRT2 deacetylate FOXO factors to regulate FOXO function. Because acetylation and ubiquitination both modify the ɛ-amino group of lysine residues, we investigated whether FOXO3 deacetylation by SIRT1 or SIRT2 facilitates FOXO3 ubiquitination and subsequent proteasomal degradation. We found that SIRT1 and SIRT2 promote FOXO3 poly-ubiquitination and degradation. Proteasome-inhibitor treatment prevented sirtuin-induced FOXO3 degradation, indicating that this process is proteasome dependent. In addition, we demonstrated that E3 ubiquitin ligase subunit Skp2 binds preferentially to deacetylated FOXO3. Overexpression of Skp2 caused poly-ubiquitination of FOXO3 and degradation, whereas knockdown of Skp2 increased the amount of FOXO3 protein. We also present evidence that SCF-Skp2 ubiquitinates FOXO3 directly in vitro. Furthermore, mutating four known acetylated lysine residues (K242, K259, K290 and K569) of FOXO3 into arginines to mimic deacetylated FOXO3 resulted in enhanced Skp2 binding but with inhibition of FOXO3 ubiquitination; this suggests that some or all of these four lysine residues are likely the sites for ubiquitination. In the livers of mice deficient in SIRT1, we detected increased expression of FOXO3, indicating SIRT1 regulates FOXO3 protein levels in vivo. Furthermore, we found that the elevation of SIRT1 and Skp2 expression in malignant PC3 and DU145 prostate cells is responsible for the downregulation of FOXO3 protein levels in these cells. Taken together, our data support the notion that deacetylation of FOXO3 by SIRT1 or SIRT2 facilitates Skp2-mediated FOXO3 poly-ubiquitination and proteasomal degradation.