Prostate cancer is the most commonly diagnosed tumor in men in the United States. Patients with hormone-refractory prostate cancer are often treated with paclitaxel, but most of them eventually develop drug resistance. S-phase kinase associated protein 2 (Skp2) is a component of the SCF (Skp1-Cullin1-F-box) type of E3 ubiquitin ligase complexes. In the present study, we investigated the role of Skp2 in paclitaxel-resistant DU145-TxR or PC-3-TxR cells by Skp2 silencing or using Skp2 inhibitors. We first confirmed that Skp2 expression is up-regulated in DU145-TxR or PC-3-TxR cells compared with their parental cells DU145 or PC-3, respectively. Knockdown of Skp2 or Skp2 inhibitor treatment in DU145-TxR or PC-3-TxR cells restored paclitaxel sensitivity. E-cadherin was decreased while Vimentin was increased in PC-3-TxR or DU145-TxR cells. In addition, p27 expression was inversely correlated with Skp2 expression in DU145-TxR or PC-3-TxR cells. Moreover, p27 was found to increase in both Skp2 silencing PC-3-TxR and DU145-TxR cells. These results suggest that Skp2 is associated with prostate cancer cell resistance to paclitaxel. Skp2 may be a potential therapeutic target for drug-resistant prostate cancer.