B7-H3 and B7x are recently discovered members of the B7-CD28 family thought to dampen peripheral immune responses via negative costimulation. We evaluated their potential expression in human prostate cancer using a large cohort of patients with 7 years of follow-up. We identified 823 patients with tissue available treated with radical prostatectomy between 1985 and 2003. Immunohistochemistry was performed on tissue microarray sections using anti-B7-H3 and -B7x. The percentage and intensity of immunoreactivity by tumor cells were blindly evaluated by two urological pathologists, and outcome analyses were conducted. Both B7-H3 and B7x were highly expressed; 93% and 99% of tumors had aberrant expression, respectively. The median percentage of tumor cells staining positive was 80% for each molecule. Strong intensity for B7-H3 and B7x was noted in 212 (26%) and 120 (15%) patients, respectively. Patients with strong intensity for B7-H3 and B7x were significantly more likely to have disease spread at time of surgery (P < 0.001 and P = 0.005, respectively). Additionally, patients with strong intensity for B7-H3 and B7x were at significantly increased risk of clinical cancer recurrence (P < 0.001 and P = 0.005) and cancer-specific death (P = 0.004 and P = 0.04, respectively). To our knowledge, we present the largest investigation of B7 family molecules in a human malignancy and a previously undescribed evaluation of B7x in prostate cancer. B7-H3 and B7x are abundantly expressed in prostate cancer and associated with disease spread and poor outcome. Given the proposed immune-inhibitory mechanisms of B7-H3 and B7x, these molecules represent attractive targets for therapeutic manipulation in prostate cancer.