Objective: To evaluate the effects of diet-induced changes in energy balance and body weight on in vivo colonocyte fractional proliferation rates (FPR) in people with obesity. Methods: In vivo colonocyte FPR was assessed in 31 men and women with obesity (BMI: 35.4 ± 4.0 kg/m2, age: 52.6 ± 8.9 years) before and after diet-induced weight loss, weight gain, or weight maintenance. Subjects ingested aliquots of 2H2O (heavy water) daily for 4 to 7 days, followed by flexible sigmoidoscopy with colon biopsies to assess the incorporation of 2H into the DNA of dividing colonocytes. Results: Colonocyte FPR averaged 12.7% ± 3.8% per day and correlated directly with intra-abdominal adipose tissue (IAAT) volume (r = 0.364, P = 0.044). Colonocyte FPR decreased in the weight loss group, did not change in the weight maintenance group, and increased in the weight gain group. The change in colonocyte FPR correlated directly with the percent change in body weight (r = 0.409, P = 0.028) and IAAT volume (r = 0.598, P = 0.001). Conclusions: A high-calorie diet and weight gain increase, whereas a low-calorie diet and weight loss decrease, in vivo colonocyte proliferation rate in people with obesity. These results suggest that changes in energy balance influence the risk of developing colon cancer in people with obesity by regulating colonic mucosal growth rates.