Abstract Background: Diet and the gut microbiome have a complex interaction that generates metabolites with an unclear effect on lethal prostate cancer risk. Identification of modifiable risk factors for lethal prostate cancer is challenging given the long natural history of this disease and difficulty of prospectively identifying lethal cancers. Methods: Mass spectrometry was performed on baseline serum samples collected from 173 lethal prostate cancer cases and 519 controls enrolled in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening trial. Baseline serum levels of choline, carnitine, betaine, γ-butyrobetaine, crotonobetaine, phenylacetylglutamine, hippuric acid, and p-cresol sulfate were quantified and analyzed by quartile. Conditional multivariable logistic regression analysis associated analyte levels with lethal prostate cancer incidence after adjusting for body mass index and PSA. The Cochran–Armitage test evaluated analyte level trends across quartiles. Results: Relative to those in the first quartile, cases with the highest baseline levels of choline (Q4 OR: 2.19; 95% CI, 1.23–3.90; P-trend: 0.005) and betaine (Q4 OR: 1.86; 95% CI, 1.05–3.30; P-trend: 0.11) exhibited increased odds of developing lethal prostate cancer. Higher baseline serum levels of phenylacetylglutamine (Q4 OR: 2.55; 95% CI, 1.40–4.64; P-trend: 0.003), a gut microbiome metabolite of phenylalanine with adrenergic activity, were also associated with lethal prostate cancer. Conclusions: Baseline serum levels of one-carbon methyl donors and adrenergic compounds resulting from human and gut microbiota–mediated metabolism are associated with increased lethal prostate cancer risk. Impact: Dietary composition, circulating metabolite levels, and downstream signaling pathways may represent modifiable risk factors associated with incident lethal prostate cancer. Beta-adrenergic blockade represents an additional target for oncologic risk reduction.