The natural isotopic composition of ¹³C and ¹²C in tissues is largely determined by the diet. Sources of provitamin A carotenoids (e.g., vegetables) typically have a lower ¹³C to ¹²C ratio (¹³C:¹²C) than preformed vitamin A sources (i.e., dairy and meat) from corn-fed animals, which are prevalent in the US. The ¹³C:¹²C of serum retinol (¹³C:¹²C-retinol) was evaluated as a biomarker for vegetable intake in a 3-mo dietary intervention designed to promote weight-loss by increased vegetable consumption or reduced calorie and fat intake. Subjects were 21–50 y of age with a BMI between 30–40 kg/m² and were enrolled from one geographic area in the US. The high vegetable group ( n = 20) was encouraged to increase daily vegetable and fruit consumption to 0.95 liter vegetables and 0.24–0.35 liter fruits. The caloric reduction group ( n = 17) was encouraged to lower caloric intake by 500 kcal and consume ≤25% kcal from fat daily. Provided meals supplied 75–100% vegetable and fruit goals and 50–67% kcal and fat g per day. Carotenoid supplementation was discontinued by subjects during the study. Serum retinol and provitamin A carotenoid concentrations; intake of preformed vitamin A, provitamin A, and fat; and body weight, fat mass, and lean mass were analyzed for correlations to ¹³C:¹²C-retinol. ¹³C:¹²C-Retinol decreased in the vegetable group after intervention ( P = 0.050) and the correlation with provitamin A intake was approaching significance ( P = 0.079). ¹³C:¹²C-Retinol did not change in the caloric reduction group ( P = 0.43). ¹³C:¹²C-Retinol changes with the vitamin A source in the diet and can be used as a biomarker for increases in dietary provitamin A vegetable intake.