Men cycle more than women and gender differences in perceptions and attitudes towards cycling may be influenced by such difference in bicycle use. However, to our knowledge, no previous research has focused on gender differences among regular cyclists. In our study, we investigated gender differences in attitudes towards cycling and towards cycling infrastructure, purpose of cycling, risk perception, and exposure to severe crashes in a large sample of regular cyclists. Following a cross-sectional design, we collected data from 2417 participants from Hungary, Italy, Spain, Sweden, Netherlands, and United Kingdom. A survey was administered to an online panel of respondents. Gender differences in attitudes towards cycling were small in terms of effect size or non-significant, with women having more positive attitudes in personal benefits rather than mobility benefits. Women reported gender-stereotyped reasons for cycling more than men, except for social activities. Also, women showed higher discomfort than men cycling in mixed traffic and higher risk perception than men. Furthermore, men reported higher exposure to severe crashes than women. We contend that bicycle use and gender role (i.e. society's shared beliefs concerning a range of attitudes, norms, and behaviours that are generally considered appropriate or desirable for individuals based on their actual or perceived sex) can affect differences between male and female cyclists in perceptions, attitudes towards cycling, and cycling behaviours.