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Regular physical activity prevents and treats cancer patients by assisting and improving the immune system. Co-stimulatory molecules that activate the immune system have been studied in cancer, such as immune checkpoint molecules of the CD40/CD40L pathway. This study aimed to characterize plasma levels of soluble CD40 (sCD40) and CD40 ligand (sCD40L) in older people with gastrointestinal tract (GIT) cancer and associate results with physical activity. This prospective and exploratory cohort study was performed with 24 older people with GIT cancer and 23 healthy elderly individuals as controls. Physical activity level was classified as active or sedentary according to the International Physical Activity Questionnaire-Short Form (IPAQ-SF). Plasma levels of sCD40 and sCD40L were determined using Enzyme-Linked Immunosorbent Assay. Plasma levels of sCD40 were higher, while sCD40L were lower (p = 0.0171) in older people with GIT cancer than controls (p = 0.0038). Regarding physical activity, active older people with GIT cancer presented lower plasma levels of sCD40 and sCD40L than those sedentary with GIT cancer (p = 0.0228 and p = 0.0236), respectively. Our findings suggest that GIT cancer stimulates the immune system in older people, elevates levels of sCD40, and reduces levels of sCD40L. Physical activity may be a protective factor for the immune system of these patients since it acts on sCD40/sCD40L pathway.