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Transport planning strategies regard cycling promotion as a suitable means for tackling problems connected with motorized traffic such as limited space, congestion, and pollution. However, the evidence base for optimizing cycling promotion is weak in most cases, and information on bicycle patterns at a sufficient resolution is largely lacking. In this paper, we propose agent-based modeling to simulate bicycle traffic flows at a regional scale level for an entire day. The feasibility of the model is demonstrated in a use case in the Salzburg region, Austria. The simulation results in distinct spatio-temporal bicycle traffic patterns at high spatial (road segments) and temporal (minute) resolution. Scenario analysis positively assesses the model’s level of complexity, where the demographically parametrized behavior of cyclists outperforms stochastic null models. Validation with reference data from three sources shows a high correlation between simulated and observed bicycle traffic, where the predictive power is primarily related to the quality of the input and validation data. In conclusion, the implemented agent-based model successfully simulates bicycle patterns of 186,000 inhabitants within a reasonable time. This spatially explicit approach of modeling individual mobility behavior opens new opportunities for evidence-based planning and decision making in the wide field of cycling promotion