Nobiletin as a nontoxic dietary citrus flavonoid has anticancer effects in cancer. Toll-like receptor three has a role in prostate cancer progression. However the relationship among NOB and TLR3 signaling in PCa has not been elucidated, yet. Therefore, we aimed to evaluate the effects of NOB on the activation of TLR3 signaling pathways in PCa In Vitro. PC-3, LNCaP and HUVEC cells were used for comparison of NOB-mediated TLR3 signaling pathways. After treatment with NOB and Poly I:C alone and NOB + Poly I:C, RT-PCR, western blotting and ELISA assay were performed to evaluate changes in gene and protein expression level, as well as CASP8. NOB potentially induced TLR3/IRF3 signaling pathway and the activation of TLR3/IRF3 signaling pathway by both NOB and Poly I:C was more profound in LNCaP than PC-3 cells. However, the level of TRIF protein and CASP8 decreased after both NOB and Poly I:C incubation. NOB could mediate TLR3 signaling pathways. NOB + Poly I:C could improve the activation of TLR3/IRF3 signaling pathway. However, the activation of TRIF/RIPK1/FADD signaling pathway reduced. Therefore, the elucidation of molecular mechanisms of TLR3 signaling pathways and the combination effects of NOB + Poly I:C on apoptotic cell death are further studied.