Abstract Pre-exposure prophylaxis (PrEP) use prevents HIV transmission, and may lead to changes in sexual behavior. We aimed to explore sexual behavior over time, and identify predictors of behavior change in men who have sex with men (MSM) using PrEP at sexual health centers (SHC) in the Netherlands. We used longitudinal data from the national STI surveillance database (January 2018–June 2021) of HIV-negative MSM who first initiated PrEP in the national PrEP pilot. We modelled behavior change after PrEP initiation, and identified predictors of behavior change using multi-state Markov models. The probability of stopping group sex, chemsex, use of poppers or erection stimulants, and having ≥ 10 partners in the past 6 months among included MSM (n = 4,349, n SHC visits = 21,820) was higher compared to the probability of starting with these behaviors after PrEP initiation. However, MSM who used condoms consistently during anal sex had a high probability of changing to inconsistent condom use (0.8) at the next visit, and inconsistent condom users mostly remained inconsistent (0.8). First visit, visiting the SHC more often (vs. regular), and STI diagnosis were predictors of starting or continuing with most of the behaviors associated with increased risk. Behavior change was less likely among older participants (> 34 compared to ≤ 34 years), and during COVID-19 lockdown and post-lockdown periods compared to pre-COVID-19. Although condom use decreased over time, transitions towards stopping with other behaviors associated with increased likelihood of acquiring an STI after PrEP initiation were common. This may suggest increased sexual empowerment, especially among younger MSM. Predictors of behavior change may help to identify MSM who are likely to start with or continue to engage in these behaviors in the near future and to provide suitable and timely counselling about behavior and PrEP adherence.