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Trans and non-binary individuals are particularly vulnerable to discrimination in public spaces, workplaces, housing, and healthcare. The exclusion they experience also affects their participation in physical activity (PA). This study aimed to estimate the proportion of trans and non-binary individuals living in Spain that engage in PA several times per week and to explore potential associated factors. A cross-sectional study was conducted using data from 1473 participants aged 15 or older, recruited between October 2023 and March 2024 through an online national survey on trans and non-binary health. PA engagement was estimated with 95% confidence intervals (95% CIs). A Poisson regression model with robust variance was used to identify associated factors, obtaining adjusted prevalence ratios (aPRs) with 95% CI. Overall, 23.2% (95% CI: 21.1%-25.5%) reported engaging in PA several times per week. In the adjusted analysis, PA was less common among trans women (aPR = 0.46, 95% CI 0.35-0.62) and non-binary individuals (aPR = 0.67, 95% CI 0.52-0.85) compared to transmasculine individuals. Lower PA levels were also observed in those unable to legally change their name due to barriers (aPR = 0.71, 95% CI 0.52-0.97), and those with mental health issues in the past year (aPR = 0.76, 95% CI 0.62-0.93). Higher PA participation was associated with being ≥29 years old (aPR = 1.41, 95% CI 1.08-1.84) and better self-perceived health (aPR = 1.33, 95% CI 1.07-1.65). Trans women and non-binary individuals engage in PA less frequently. Sociodemographic factors, health, and legal barriers significantly impact PA participation. Policies should encourage PA, especially among young trans individuals, and ensure equitable and discrimination-free access.