STUDY QUESTION: Are use of e-cigarettes and snuff associated with testicular function as previously shown for conventional cigarettes and marijuana? SUMMARY ANSWER: Use of e-cigarettes is associated with reduced semen quality but not with higher serum testosterone level as observed for conventional cigarette use. Snuff use was not associated with markers of testicular function. WHAT IS KNOWN ALREADY: Cigarette smoking has previously been associated with higher testosterone levels and impaired semen quality, whereas it is unresolved whether use of e-cigarettes or snuff influence the testicular function. STUDY DESIGN, SIZE, DURATION: This cross-sectional population-based study included 2008 men with information on cigarette and marijuana use (enrolled between 2012 and 2018), among whom 1221 men also had information on e-cigarette and snuff use (enrolled between 2015 and 2018). PARTICIPANTS/MATERIALS, SETTING, METHODS: Men (median age 19.0 years) from the general population provided a semen and blood sample and filled out a questionnaire on lifestyle including information on smoking behaviour. Associations between different types of smoking (e-cigarettes, snuff, marijuana and cigarettes) and reproductive hormones (total and free testosterone, sex hormone-binding globulin, LH, oestradiol and ratios of inhibin B/FSH, testosterone/LH and free testosterone/LH) and semen parameters (total sperm count and sperm concentration) were examined using multiple linear regression analyses adjusted for relevant confounders. MAIN RESULTS AND THE ROLE OF CHANCE: Approximately half of the men (52%) were cigarette smokers, 13% used e-cigarettes, 25% used snuff and 33% used marijuana. Users of e-cigarettes and marijuana were often also cigarette smokers. Compared to non-users, daily e-cigarette users had significantly lower total sperm count (147 million vs 91 million) as did daily cigarette smokers (139 million vs 103 million), in adjusted analyses. Furthermore, significantly higher total and free testosterone levels were seen in cigarette smoking men (6.2% and 4.1% higher total testosterone and 6.2% and 6.2% higher free testosterone in daily smokers and occasional smokers, respectively, compared to non-smoking men), but not among e-cigarette users. Daily users of marijuana had 8.3% higher total testosterone levels compared to non-users. No associations were observed for snuff in relation to markers of testicular function. LIMITATIONS, REASONS FOR CAUTION: We cannot exclude that our results can be influenced by residual confounding by behavioural factors not adjusted for. The number of daily e-cigarette users was limited and findings should be replicated in other studies. WIDER IMPLICATIONS OF THE FINDINGS: This is the first human study to indicate that not only cigarette smoking but also use of e-cigarettes is associated with lower sperm counts. This could be important knowledge for men trying to achieve a pregnancy, as e-cigarettes are often considered to be less harmful than conventional cigarette smoking. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from the Danish Ministry of Health (1-1010-308/59), the Independent Research Fund Denmark (8020-00218B), ReproUnion (20200407) and the Research Fund of the Capital Region of Denmark (A6176). The authors have nothing to disclose.NA.