Our laboratory has previously identified soluble guanylyl cyclase α1 (sGCα1) as a novel androgen-regulated gene essential for prostate cancer cell proliferation. sGCα1 expression is highly elevated in prostate tumors, contrasting with the low expression of sGCβ1, with which sGCα1 dimerizes to mediate nitric oxide (NO) signaling. In studying its mechanism of action, we have discovered that sGCα1 can inhibit the transcriptional activity of p53 in prostate cancer cells independent of either classical mediators of NO signaling or the guanylyl cyclase activity of sGCα1. Interestingly, sGCα1 inhibition of p53-regulated gene expression was gene specific, targeting genes involved in apoptosis/cell survival. Consistent with this, overexpression of sGCα1 makes prostate cancer cells more resistant to etoposide, a chemotherapeutic and apoptosis-inducing drug. Immunoprecipitation and immunocytochemistry assays show a physical and direct interaction between sGCα1 and p53 in prostate cancer cells. Interestingly, sGCα1 induces p53 cytoplasmic sequestration, representing a new mechanism of p53 inactivation in prostate cancer. Analysis of prostate tumors has shown a direct expression correlation between sGCα1 and p53. Collectively, these data suggest that sGCα1 regulation of p53 activity is important in prostate cancer biology and may represent an important mechanism of p53 down-regulation in those prostate cancers that express significant levels of p53.