Epidemiological evidence shows that regular physical activity is associated with reduced risk of primary and recurrent colon cancer. However, the underlying mechanisms of action are poorly understood. We evaluated the effects of stimulating a human colon cancer cell line (LoVo) with human serum collected before and after an acute exercise bout vs nonexercise control serum on cancer cell proliferation. We also measured exercise‐induced changes in serum cytokines and intracellular protein expression to explore potential biological mechanisms. Blood samples were collected from 16 men with lifestyle risk factors for colon cancer (age ≥50 years; body mass index ≥25 kg/m2; physically inactive) before and immediately after an acute bout of moderate‐intensity aerobic interval exercise (6 × 5 minutes intervals at 60% heart rate reserve) and a nonexercise control condition. Stimulating LoVo cells with serum obtained immediately after exercise reduced cancer cell proliferation compared to control (−5.7%; P = .002). This was accompanied by a decrease in LoVo cell γ‐H2AX expression (−24.6%; P = .029), indicating a reduction in DNA damage. Acute exercise also increased serum IL‐6 (24.6%, P = .002). Furthermore, stimulating LoVo cells with recombinant IL‐6 reduced γ‐H2AX expression (β = −22.7%; P < .001) and cell proliferation (β = −5.3%; P < .001) in a linear dose‐dependent manner, mimicking the effect of exercise. These findings suggest that the systemic responses to acute aerobic exercise inhibit colon cancer cell proliferation in vitro, and this may be driven by IL‐6‐induced regulation of DNA damage and repair. This mechanism of action may partly underlie epidemiological associations linking regular physical activity with reduced colon cancer risk.