Objective: To determine the effect of biological mesh closure on perineal wound healing after extralevator abdominoperineal resection (eAPR). Background: Perineal wound complications frequently occur after eAPR with preoperative radiotherapy for rectal cancer. Cohort studies have suggested that biological mesh closure of the pelvic floor improves perineal wound healing. Methods: Patients were randomly assigned to primary closure (standard arm) or biological mesh closure (intervention arm). A non-cross-linked porcine acellular dermal mesh was sutured to the pelvic floor remnants in the intervention arm, followed by a layered closure of the ischioanal and subcutaneous fat and skin similar to the control intervention. The outcome of the randomization was concealed from the patient and perineal wound assessor. The primary endpoint was the rate of uncomplicated perineal wound healing defined as a Southampton wound score of less than 2 at 30 days postoperatively. Patients were followed for 1 year. Results: In total, 104 patients were randomly assigned to primary closure (n = 54; 1 dropouts) and biological mesh closure (n = 50; 2 dropouts). Uncomplicated perineal wound healing rate at 30 days was 66% (33/50; 3 not evaluable) after primary closure, which did not significantly differ from 63% (30/48) after biological mesh closure [relative risk 1.056; 95% confidence interval (CI) 0.7854-1.4197; P = 0.7177). Freedom from perineal hernia at 1 year was 73% (95% CI 60.93-85.07) versus 87% (95% CI 77.49-96.51), respectively (P = 0.0316). Conclusions: Perineal wound healing after eAPR with preoperative radiotherapy for rectal cancer was not improved when using a biological mesh. A significantly lower 1-year perineal hernia rate after biological mesh closure is a promising secondary finding that needs longer follow-up to determine its clinical relevance.