This randomized controlled cross-over study aimed to assess the efficacy and safety of glans penis injection with hyaluronic acid (HA) in treating premature ejaculation (PE). A total of 30 patients with PE were randomly allocated into two groups: group 1 (n = 15) which was subjected to glans penis HA injection and group 2 (n = 15) which was injected with saline as a control then both groups were subjected to follow-up at 1 week and 1 month after injection. These subjects were evaluated by intra-vaginal ejaculation latency time (IELT) and the Arabic validated index of premature ejaculation (AIPE). After a wash-out period, cross-over and re-evaluation of both groups were carried. Additionally, patients with reported improvement after 1 month of HA injection (n = 20) were subjected to extended evaluation by IELT at 3, 6, and 9 months intervals. Two-way repeated measures ANOVA indicated significant improvement after HA in comparison with saline across the follow-up periods (F(1.66: 91.37) = 24.85, p = 0.001). Post-hoc Bonferroni test indicated no significant difference after 1 week period in comparison with baseline IELT, but a significant difference after 1 month of injection in comparison with baseline IELT (p < 0.001) and after 1 week (p < 0.001). After 1 month of HA, IELT increased by a median of 2.6 folds while 1.1 folds increase was observed after 1 month of saline injection. Total AIPE scores improved significantly after HA injection compared with baseline (p = 0.003) and saline scores (p = 0.002). Reported adverse effects were minimal and self-limited. It could be concluded that glans penis injection with HA for treatment of PE is a safe method that ensures a modest but significant increase in IELT and improves couple sexual satisfaction.