Pre-exposure prophylaxis (PrEP) use may influence sexual behaviour and transmission of STIs among men who have sex with men (MSM). We aimed to quantify the distribution of STI diagnoses among MSM in the Netherlands based on their sexual behaviour before and after the introduction of PrEP. HIV-negative MSM participating in a prospective cohort study (Amsterdam Cohort Studies) completed questionnaires about sexual behaviour and were tested for STI/HIV during biannual visits (2009-2019). We developed a sexual behaviour risk score predictive of STI diagnosis and used it to calculate Gini coefficients for gonorrhoea, chlamydia and syphilis diagnoses in the period before (2009 to mid-2015) and after PrEP (mid-2015 to 2019). Gini coefficients close to zero indicate that STI diagnoses are homogeneously distributed over the population, and close to one indicate that STI diagnoses are concentrated in individuals with a higher risk score. The sexual behaviour risk score (n=630, n visits=10 677) ranged between 0.00 (low risk) and 3.61 (high risk), and the mean risk score increased from 0.70 (SD=0.66) before to 0.93 (SD=0.80) after PrEP. Positivity rates for chlamydia (4%) and syphilis (1%) remained relatively stable, but the positivity rate for gonorrhoea increased from 4% before to 6% after PrEP. Gini coefficients increased from 0.37 (95% CI 0.30 to 0.43) to 0.43 (95% CI 0.36 to 0.49) for chlamydia, and from 0.37 (95% CI 0.19 to 0.52) to 0.50 (95% CI 0.32 to 0.66) for syphilis comparing before to after PrEP. The Gini coefficient for gonorrhoea remained stable at 0.46 (95% CI 0.40 to 0.52) before and after PrEP. MSM engaged in more high-risk sexual behaviour and gonorrhoea diagnoses increased after PrEP was introduced. Chlamydia and syphilis diagnoses have become more concentrated in a high-risk subgroup. Monitoring the impact of increasing PrEP coverage on sexual behaviour and STI incidence is important. Improved STI prevention is needed, especially for high-risk MSM.