Physical activity is an important factor in attaining bone mass. Our aim was to investigate if low to moderate intensity exercise affects bone resorption [serum tartrate-resistant acid phosphatase (TRAP) 5b activity] and formation (serum osteocalcin concentration) in a randomized controlled exercise intervention trial in Finnish middle-aged men. In addition, the relations of these bone turnover markers with bone mineral density (BMD) and serum sex hormone concentrations [circulating testosterone (T), estradiol (E2), and sex hormone-binding globulin (SHBG) concentrations] were evaluated. Serum TRAP 5b activity and osteocalcin concentration were measured at randomization and after 1 and 4 years of the exercise intervention. BMDs of the lumbar spine (L2-L4), femoral neck, and total proximal femur were measured with a dual-energy X-ray absorptiometry (DXA). At randomization, TRAP 5b activity was strongly correlated with the osteocalcin concentration (Spearman r = 0.541, P < 0.0001). In addition, TRAP 5b activity was significantly correlated with proximal femur BMD values (r = -0.201, P = 0.018) and osteocalcin concentration with femoral neck and proximal femur BMD values (r = -0.187, P = 0.028; r = -0.240, P = 0.005, respectively). Serum E2, free E2, and free T concentrations were inversely correlated with both bone turnover markers. After 1 year of exercise intervention, TRAP 5b activity was significantly lower in the exercise than reference group (P = 0.006). However, after 4 years of exercise intervention, the difference was no longer statistically significant. There were no differences in the osteocalcin concentrations between the study groups during the intervention. Our results show a connection between serum TRAP 5b activity and osteocalcin concentration. Furthermore, our results suggest that low to moderate exercise intervention and serum sex hormone concentrations may induce changes in bone metabolism in middle-aged men. However, exercise-induced effects on bone metabolism should be confirmed in other randomized controlled exercise trials taking into account exercise intensity and dose-response issues.