No human study has investigated the possible impact of breastfeeding on semen quality and levels of reproductive hormones, but a recent study of another hypothesis indicated an association with oligozoospermia. We investigated the association between breastfeeding, semen quality and levels of reproductive hormones. From a Danish pregnancy cohort established in 1984–1987, 347 sons were selected according to maternal smoking during pregnancy and followed up with questionnaires, semen analysis and blood sampling in 2005–2006. Complete data were available for 269 men aged 18–21 years. Breastfeeding was not statistically significantly associated with sperm concentration, total sperm count, sperm motility or morphology, oligozoospermia, follicle-stimulating hormone, inhibin B, luteinizing hormone, sex hormone-binding globulin (SHBG), the calculated level of free testosterone, free oestradiol, the free testosterone/free oestradiol ratio or the follicle-stimulating hormone/inhibin B ratio. Total testosterone and total oestradiol was 16% (p = 0.01) and 14% (p = 0.06), respectively, lower among men never breastfed in comparison with men breastfed exclusively for 1 month or longer. When taking SHBG into account, neither free testosterone nor free oestradiol was different between the two groups. This study shows no association between breastfeeding and sperm quality or reproductive hormones and a strong association is unlikely. A larger study would be needed to detect more subtle effects.