Objective: To investigate the influences of using methylene blue injection into the superior rectal artery during laparoscopic rectal resection after neoadjuvant chemoradiotherapy on number of harvested lymph nodes and postoperative male sexual function and urination function in male rectal cancer patients. Methods: Ninety-six rectal cancer male patients undergoing laparoscopic radical resection after synchronous neoadjuvant chemotherapy and radiotherapy in Xiangtan Central Hospital and Chen Xinghai Hospital during March 2013 to April 2017 were enrolled. The patients were randomly assigned to study group and control group, with 48 cases in each group. Patients in study group were injected with 8 mL of 1% methylene blue into the superior rectal artery prior to radical resection, and those in control group underwent radical surgery directly. The numbers of lymph node dissected, and postoperative sexual function (including erection and ejaculation) and urination function between the two groups of patients were compared. Results: There was no significant difference in the procedures performed in the two groups (P>0.05). The number of lymph nodes removed was significantly higher in study group than that in control group (15.04±4.063 vs. 12.23±2.991, P<0.05); the numbers of cases with grade I, II and III postoperative 1-year ejaculation function were 41 (85.4%), 7 (14.6%) and 0 (0) in study group, and were 31 (64.6%), 13 (27.1%) and 4 (8.3%) in control group, the numbers of cases with grade I, II, III postoperative 1-year erection function were 42 (87.5%), 6 (12.5%) and 0 (0) in study group and were 32 (66.7%), 15 (31.3%) and 1 (2.1%) in control group, and the numbers of cases with grade I, II, III and IV postoperative 1-year urination function were 35 (72.9%), 12 (25.0%) , 1 (2.1%) and 0 (0) in study group and were 26 (54.2), 13 (27.1%), 5 (10.4%) and 4 (8.3%) in control group. The results of statistical analysis showed the postoperative 1-year ejaculation function, erection function and urination function were significantly superior in study group than those in control group (all P<0.05). Conclusion: For rectal cancer patients undergoing radical resection after neoadjuvant chemoradiotherapy, methylene blue injection into the superior rectal artery can help to clarify the surgical plane and increase the lymph node dissection rate as well as reduce damages of the pelvic nerves, and thereby protect the sexual function and urinary function of male patients.