Purpose: To evaluate the association between ejaculation frequency (EF) during four stages of life and prostate cancer (PCa) according to tumor aggressiveness, PCa stage, and urinary symptomatology. Materials and Methods: A total of 456 incident PCa cases histologically confirmed, and 427 controls aged 40–80 years from the CAPLIFE study were analyzed. This study is a population-based case-control study carried out in the south of Spain. Average EF was measured for: (1) 20s, (2) 30s, (3) 40s, and (4) one year before the interview. EF was categorized into: (1) 0–3, (2) 4, and (3) >4 ejaculations/month. Sociodemographic, lifestyle, and medical information were also collected. To estimate the association between EF and PCa, adjusted ORs (aORs) and 95% CIs were calculated by logistic regression models. Results: A year before the interview, PCa cases ejaculated less frequently than the controls. An inverse association was observed between the EF a year before and PCa, aOR=1.64 (95% CI 1.03–2.61) for men with 4 ejaculations/month, and aOR=2.38 (95% CI 1.57–3.60) for men with 0–3 ejaculations/month, compared to men with >4. The association was higher for cases with ISUP 3–5 (aOR=2.76 [95% CI 1.34–5.67] for men with 0–3 ejaculations/month) or with a locally advanced-metastatic tumor (aOR=4.70 [95% CI 1.55–14.29]). Moreover, men with moderate urinary symptoms and 0–3 ejaculations/month had the highest risk, aOR=3.83 (95% CI 1.84–7.95). Conclusions A low EF could be associated with a higher risk of PCa, especially for cases with ISUP 3–5 or with a locally advanced-metastatic tumor.