ABSTRACT Objective: To assess safety and efficacy of hyaluronic acid (HA) gel injection in glans penis for treatment of premature ejaculation (PE) using our new five puncture technique. Patients and methods: This is a prospective, non-randomized clinical trial on HA gel injection in glans penis for all patients with lifelong PE; all patients were circumcised having heterosexual normal marital life and sexually active. Patients with history of ejaculatory medication use within the previous 3 months, psychiatric disorders, erectile dysfunction, lower urinary tract symptoms (LUTS) due to prostatitis and acquired PE were excluded from the study. A local anesthetic was applied to the skin of glans penis for 30 minutes before the injection of 2 ml HA in glans penis via 30-gauge needle using our new Five-puncture technique. Intra-vaginal ejaculatory latency time (IELT) was measured at 1, 3, 6 and 12 months after injection. Results: Thirty patients completed our study follow up schedule. Mean age of the patients was 41.72 ± 8.50, while mean age of female partner was 37.23 ± 8.54 years. IELT was highly significantly increased (P-value < 0.001) after HA gel injection from baseline, which was in maximum 37.83 ± 11.01 sec at baseline to 323.03 ± 42.06, 281.07 ± 41.05, 241.03 ± 43.09 and 235.6 ± 41.87 sec after 1, 3, 6 and 12 months, respectively, after injection. Three patients complained from discomfort at the site of injection, two from bullae formation at the site of injection and one from ecchymosis, and all resolved spontaneously after 1 week to 10 days after injection. Conclusion: HA gel injection in glans penis using our new five-puncture technique is a safe and effective method that ensures a modest long-term significant increase in IELT and improves ejaculatory control.