High physical activity is associated with a lower risk of colon cancer; however, the underlying biological mechanisms for this association remain largely unclear. We aimed to determine the extent to which body fat and biomarkers of various biologically plausible pathways account for the association between physical activity and colon cancer. We conducted a nested case-control study in a cohort of 519,978 men and women aged 25 to 70 years participating in the European Prospective Investigation into Cancer and Nutrition Cohort (EPIC) followed from 1992 to 2003. A total of 713 incident colon cancer cases were matched using risk-set sampling to 713 controls on age, sex, study center, fasting status, and hormonal therapy use. The amount of total physical activity during the past year was expressed in metabolic equivalent of task [MET]-hours/week. Anthropometric measurements and blood samples were collected at study baseline. Biomarkers in the following pathways were evaluated: metabolic dysfunction, insulin growth factors, adipokine secretion [adiponectin, leptin, soluble leptin receptor (sOB-R)], lipid metabolism, oxidative stress, vitamin D metabolism [25[OH]D], inflammation and immune response. High physical activity was associated with a lower risk of colon cancer (Relative Risk ≥91 MET-hours/week versus < 91 MET-hours/ week = 0.75 [95% confidence interval (CI): 0.57 to 0.96]. In mediation analyses, this association was accounted for by waist circumference [proportion explained effect (PEE) = 17%; CI: 4% to 52%] and the biomarkers: soluble leptin receptor (sOB-R) [PEE = 15%; 95% CI: 1% to 50%] and 5-hydroxyvitamin D (25[OH]D) [PEE = 30%; 95% CI: 12% to 88%]. In combination, these factors explained 45% (95% CI: 20% to 125%) of the association. Beyond waist circumference, sOB-R and 25[OH]D additionally explained 10% and 23% of the association, respectively. Promoting physical activity and maintaining metabolic health and adequate vitamin D levels could represent a promising strategy for colon cancer prevention.