PrEP uptake in the Netherlands is growing but remains at suboptimal levels. Hence, the analysis of hurdles is paramount. Given the initial focus of PrEP provision among men-who-have-sex-with-men (MSM) via a demonstration project that was launched in June 2015, AmPrEP in Amsterdam, and pharmacies in the main urban areas (so called “Randstad”, entailing Amsterdam, Utrecht, Leiden, The Hague and Rotterdam), investigating regional differences is necessary. This study seeks to unravel regional differences jointly with the psycho-social determinants of PrEP uptake. This cross-sectional study included 3232 HIV-negative MSM recruited via the Dutch subsample of the European-MSM-Internet-Survey in late 2017 (EMIS-2017), which aimed to inform interventions for MSM who are highly affected by infections with HIV and other sexually transmitted infections. Prevalence and the standardised prevalence ratio (SPR) of PrEP awareness, intention and uptake were measured on a regional level (Randstad vs. the rest of the country). Multi-level logistic modelling was conducted to identify the association of PrEP uptake with PrEP awareness and intention, socio-demographic, psycho-social determinants and random effects from regional differences. MSM from the Randstad used more PrEP (SPR = 1.4 vs. 0.7) compared to the rest of the country, but there were minor differences for awareness and intention. The regional distinction was estimated to explain 4.6% of the PrEP use variance. We observed a greater influence from PrEP intention (aOR = 4.5, 95% CI 2.0–10.1), while there was limited influence from the awareness of PrEP (aOR = 0.4, 95% CI 0.04–4.4). Lower education (aOR = 0.4, 95% CI 0.2–0.9) was negatively associated with PrEP uptake; however, no significant difference was found between middle (aOR = 1.2, 95% CI 0.7–2.0) and high education. We showed that regional differences—MSM in non-urban regions—and other psycho-social determinants account for lower PrEP uptake. Based on these findings, more fine-tuned PrEP access with a focus on ...