The association between last 5 days of alcohol intake, semen quality and reproductive hormones was estimated in this cross-sectional study among 347 men. Conventional semen characteristics, DNA fragmentation index and reproductive hormones (testosterone, estradiol, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH) and inhibin B) were determined. There was a tendency towards lower semen characteristics at higher intake of alcohol past 5 days, albeit with no statistically significant dose-response association. The ratio between free estradiol and free testosterone was higher at higher alcohol intake during the 5 days preceding semen sampling. In conclusion, alcohol intake was associated with impairment of most semen characteristics but without a coherent dose-response pattern. The study indicates an association between recent alcohol intake and a hormonal shift towards higher estradiol/testosterone ratio. The hormonal changes observed may over time, lead to adverse effects on semen quality, but longitudinal studies are needed to study this.