Background: Several prospective studies suggest that use of cholesterol-lowering statin drugs is inversely associated with advanced stage and possibly high-grade prostate cancer. One study reported that men with low cholesterol had a lower risk of high-grade prostate cancer. Given these findings, we investigated the association between low serum cholesterol and prostate cancer risk in the Prostate Cancer Prevention Trial. Methods: We conducted a cohort study of 5,586 men ages ≥55 years who were randomized to the placebo arm of the Prostate Cancer Prevention Trial between 1993 and 1996. Serum cholesterol was measured enzymatically at entry. By the end of follow-up, 1,251 prostate cancer cases were confirmed. We used logistic regression to calculate the multivariable odds ratio (OR) of total, and Gleason 2 to 6 (n = 993), 7 (n = 199), and 8 to 10 (n = 59) prostate cancer comparing low serum (normal, <200 mg/dL) to high-serum (borderline and elevated cholesterol, ≥200 mg/dL) cholesterol. Results: Men with low cholesterol had a lower risk of Gleason 8 to 10 prostate cancer [OR, 0.41; 95% confidence interval (CI), 0.22-0.77] than men with high cholesterol. No association was present for prostate cancer overall (OR, 0.97; 95% CI, 0.85-1.11), Gleason 2 to 6 disease (OR, 1.03; 95% CI, 0.89-1.18), or Gleason 7 disease (OR, 0.93; 95% CI, 0.69-1.24). Conclusion: These prospective results support that men with low cholesterol have a reduced risk of high-grade prostate cancer. These and other contemporary data that suggest that cholesterol metabolism should be investigated further in the etiology of prostate cancer.