Overexpression of helix-loop-helix protein-Id1 in prostate cancer correlates with tumor differentiation, and may play a key role in the development of prostate cancer. Hence, we inactivated the Id1 gene in prostate cancer cells in vitro and in vivo to determine whether the Id1 gene has therapeutic potential in the treatment of prostate cancer. A modified small interfering RNA (siRNA) was used to inactivate the Id1 gene in androgen-independent prostate cancer cell line PC3 and its xenografts in nude mice. Downregulation of Id1 by siRNA was confirmed by real-time PCR. The changes of cell viability, apoptosis and senescence rate in PC3 were individually detected. The mRNA and protein expression of Id1, PCNA and MMP2 in xenografted tumors was further individually assayed by real-time PCR and immunohistochemistry. The mRNA and protein expression of Id1 were obviously inhibited by the siRNA strategy in PC3 cells and their xenografts (P<0.01). The cell viability of PC3 was suppressed obviously (P<0.01); meanwhile, the apoptosis and senescence rates of PC3 were significantly increased by siRNA (P<0.01). Moreover, tumor growth was inhibited by the gene silencing of Id1. Two genes involved in proliferation (PCNA) and tumor invasion (MMP2) were found significantly decreased by siRNA in PC3 xenografts (P<0.01). Our results show that inactivation of Id1 can suppress cell proliferation, induce apoptosis and senescence in PC3. Silencing of the Id1 gene has an in-vivo preventive effect against the development of prostate cancer in a mouse model.