STUDY QUESTION How are temporal trends in lifestyle factors, including exposure to maternal smoking in utero, associated to semen quality in young men from the general population? SUMMARY ANSWER Exposure to maternal smoking was associated with lower sperm counts but no overall increase in sperm counts was observed during the study period despite a decrease in this exposure. WHAT IS KNOWN ALREADY Meta-analyses suggest a continuous decline in semen quality but few studies have investigated temporal trends in unselected populations recruited and analysed with the same protocol over a long period and none have studied simultaneous trends in lifestyle factors. STUDY DESIGN, SIZE, DURATION Cross-sectional population-based study including ∼300 participants per year (total number = 6386) between 1996 and 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS The study is based on men from the Greater Copenhagen area, Denmark, with a median age of 19 years, and unselected with regard to fertility status and semen quality. The men delivered a semen sample, had a blood sample drawn and a physical examination performed and answered a comprehensive questionnaire, including information on lifestyle and the mother's pregnancy. Temporal trends in semen quality and lifestyle were illustrated graphically, and trends in semen parameters and the impact of prenatal and current lifestyle factors were explored in multiple regression analyses. MAIN RESULTS AND THE ROLE OF CHANCE Throughout the study period, 35% of the men had low semen quality. Overall, there were no persistent temporal trends in semen quality, testicular volume or levels of follicle-stimulating hormone over the 21 years studied. The men's alcohol intake was lowest between 2011 and 2016, whereas BMI, use of medication and smoking showed no clear temporal trends. Parental age increased, and exposure in utero to maternal smoking declined from 40% among men investigated in 1996-2000 to 18% among men investigated in 2011-2016. Exposure to maternal smoking was associated with lower sperm counts but no overall increase in sperm counts was observed despite the decrease in this exposure. LIMITATIONS, REASONS FOR CAUTION Information of current and prenatal lifestyle was obtained by self-report, and the men delivered only one semen sample each. WIDER IMPLICATIONS OF THE FINDiNGS The significant decline in in utero exposure to maternal smoking, which was not reflected in an overall improvement of semen quality at the population level, suggest that other unknown adverse factors may maintain the low semen quality among Danish men. STUDY FUNDiNG/COMPETING INTEREST(S) The study has received financial support from the ReproUnion; the Research fund of Rigshospitalet, Copenhagen University Hospital; the European Union (Contract numbers BMH4-CT96-0314,QLK4-CT-1999-01422, QLK4-CT-2002-00603, FP7/2007-2013, DEER Grant agreement no. 212844); the Danish Ministry of Health; the Danish Environmental Protection Agency; A.P. Møller and wife Chastine McKinney Møllers foundation; and Svend Andersens Foundation. None of the funders had any role in the study design, collection, analysis or interpretation of data, writing of the paper or publication decisions. TRIAL REGISTRATION NUMBER N/A.