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Background The serum natural abundance carbon isotope ratio (CIR) was recently identified as a candidate biomarker of animal protein intake in postmenopausal women. Such a biomarker would help clarify the relation between dietary protein source (plant or animal) and chronic disease risk. Objectives We aimed to evaluate the performance of the serum CIR as a biomarker of dietary protein source in a controlled feeding study of men and women of diverse age and BMI. Methods We conducted a 15-d feeding study of 100 adults (age: 18–70 y, 55% women) in Phoenix, AZ. Participants were provided individualized diets that approximated habitual food intakes. Serum was collected at the end of the feeding period for biomarker measurements. Results Median [IQR] animal protein intake was 67 g/d [55–88 g/d], which was 64% of total protein. The serum CIR was positively correlated with animal protein and inversely correlated with plant protein intake, leading to a strong correlation (r 2 = 0.76) with the dietary animal protein ratio (APR; animal/total protein). Regressing serum CIR on the APR, serum nitrogen isotope ratio (NIR), gender, age, and body weight generated an R 2 of 0.78. Following the measurement error model for predictive biomarkers, the resulting regression equation was then inverted to develop a calibrated biomarker equation for APR. Added sugars ratio (added/total sugars intake) and corn intakes also influenced the serum CIR but to a much lesser degree than the APR; variations in these intakes had only small effects on biomarker-estimated APR. Conclusions Based on our findings in this US cohort of mixed sex and age, we propose the serum CIR alongside NIR as a predictive dietary biomarker of the APR. We anticipate using this biomarker to generate calibrated estimates based on self-reported intake and ultimately to obtain more precise disease risk estimates according to dietary protein source. [ABSTRACT FROM AUTHOR]