Background: The association between endocrine control and ejaculatory reflex is still not completely elucidated. Serum dihydrotestosterone (DHT) concentrations have been reported to be lower in premature ejaculation (PE) patients. We hypothesized that homeostasis between testosterone (T) and their metabolites; dihydrotestosterone (DHT) and estradiol (E2); could be involved in orchestration of the ejaculatory process. Aim: To test this hypothesis, we aimed to investigate any possible relationship between DHT/ E2 ratio and T/E2 ratio in a cohort of patients consulting for premature ejaculation (PE). Methods: This prospective hospital-based cross-sectional case-control study has been carried on 104 PE patients (30 patients with primary lifelong PE and 74 patients with acquired PE) compared to 90 healthy men as controls. Results: DHT levels were significantly higher in secondary PE compared to primary PE group (p = 0.011). Besides, serum E2 showed significant higher level among both primary and secondary PE groups compared to the control group (p=0.001). Moreover, both total testosterone (TT)/E2 and DHT/E2 ratios were significantly lower in both primary and secondary PE compared to the control group (p=0.001 for both). Furthermore, DHT/E2 ratio showed fair discriminating ability between PE and healthy controls (AUC = 0.749, p=0.001). Conclusions: Both TT/E2 and DHT/E2 ratios were significantly lower in both primary and secondary PE subjects suggesting a role of hormonal imbalance in this context. Although this link seems likely, large-scale studies are needed to confirm these findings.