Prostate cancer (PCa) is the second most common cancer in men. In 2012, about 300,000 men died from PCa and an estimated 1.1 million men were diagnosed with the disease world-wide. Androgens drive the progression of PCa through its activation of the androgen receptor (AR). Androgen-ablation-therapy in combination with anti-androgens are used as the mainstream treatment of PCa. However, within 18–24 months, 50% of PCas become resistant to androgen-ablation therapy, which is of major concern for the patients. Recent results in our group show that Timeless (the human homolog of a Drosophila gene involved in regulation of the circadian rhythm) interacts with and acts as a co-activator of ER alpha promoting breast cancer cell proliferation. Increased Timeless mRNA expression has been observed in PCa compared to normal tissue with the highest expression found to be in metastatic tumour tissue. To determine if Timeless acts as a co-activator of AR, we studied its effect on activity of AR (on an androgen-responsive-element-luciferase reporter construct) in the presence of the natural androgen dihydrotestosterone. Timeless increased AR activity in this model. Moreover, we show that DU145 (androgen-insensitive PCa cells) have a higher endogenous protein expression of Timeless than the androgen-sensitive LNCaP cells, indicating that Timeless may play a role in androgen-ablation resistance. Using DU145 cells, preliminary results indicate that Timeless over-expression enhances the proliferation of this cell line, and even more so in the presence of AR. We are currently determining the effects of Timeless depletion on AR activity and expression of known AR target genes, as well as on prostate cancer cell proliferation. Strategies to improve advanced PCa therapy are needed and our results indicate that Timeless may be a target for improved treatment of the patients.