Background: We performed a prospective study to evaluate the effects of pelvic irradiation on FSH, LH and testosterone levels in male patients with rectal adenocarcinoma. Our aim was to compare the level of male sex hormones in peripheral blood serum before and after pelvic irradiation.Methods: The eligible participants were 40 men with rectal adenocarcinoma who underwent pelvic radiotherapy as part of their treatment for primary tumor, either before or after surgery. All patients received a 50-Gy radiation dose to the pelvis, 2 Gy per fraction, five days per week. Blood was sampled three times during the study: once before radiation, at the end of the radiation course and 4 to 6 weeks after radiotherapy.Results: Median age of the patients was 58 years (range 18-82). The mean testis dose of radiation per fraction in all 40 patients was 16.3 cGy with a standard deviation of 15.22 (range 5.5-64.8). Serum levels of FSH revealed a significant increase from 7.5 ± 1.7 IU/L (before treatment) to 20.9 ± 17.8 IU/L [end of radiotherapy (P<0.001)] and 24.1 ± 20.5 IU/L [4 to 6 weeks after radiotherapy (P<0.001)]. Serum LH levels were significantly elevated from 8.04 ± 1.2 IU/L before radiation to 11.6 ± 11.5 IU/L at the end of radiotherapy (P<0.001) and 12.5 ± 9.9 IU/L 4 to 6 weeks after the final course of radiotherapy (P<0.001). There was a decrease in serum testosterone from 5.3±2.1 ng/mL before radiation to 4.2 ± 1.9 ng/mL at the end of radiotherapy (P=0.004) and 4.5 ± 2 ng/mL 4 to 6 weeks after radiotherapy (P=0.035). No significant correlation was seen between age and differences in sex hormones (LH, P=0.605; FSH, P=0.380; testosterone, P=0.161).Conclusion: There was a significant change in serum levels of male sex hormones after pelvic irradiation for rectal cancer (total dose, 50 Gy) that indicates considerable testicular damage under these circumstances. Thus, it seems logical to use techniques that reduce the radiation dose to the testicles and to consider the benefits of hormone replacement therapy as well as semen cryopreservation for high-risk patients who desire children in the future.