Regular exercise confers numerous physical and mental health benefits, yet individual variability in exercise participation and outcomes is still poorly understood. Uncovering the neurobiological mechanisms governing exercise behavior is essential for promoting physical activity and developing targeted interventions for related disorders. While genetic studies have provided insights, they often cannot account for protein-level alterations, such as changes in kinase activity. Here, we employ protein kinase activity profiling to delineate brain protein kinase activity and signaling networks modulated by acute voluntary exercise in rats. Focusing on the dorsal striatum, which governs voluntary exercise, as well as the hippocampus, which is susceptible to modulation by physical activity, we aim to understand the molecular basis of exercise behavior. Utilizing high throughput kinome array profiling and advanced pathway analyses, we identified protein kinase signaling pathways implicated in regulating voluntary exercise. Pathway analysis using Gene Ontology (GO) revealed significant alterations in 155 GO terms in the dorsal striatum and 206 GO terms in the hippocampus. Changes in kinase activity were observed in the striatum and hippocampus between the exercise (voluntary wheel running, VWR) and sedentary control rats. In both regions, global serine-threonine kinase (STK) activity was decreased, while global phospho-tyrosine kinase (PTK) activity was increased in VWR rats compared to control rats. We also identified specific kinases altered in VWR rats, including the IKappaB Kinase (IKK) and protein kinase delta (PKD) families. C-terminal src Kinase (CSK), epidermal growth factor (EGFR), and vascular endothelial growth factor receptor (VEGFR) tyrosine kinase were also enriched. These findings suggest regional heterogeneity of kinase activity following voluntary exercise, emphasizing potential molecular mechanisms underlying exercise behavior. This exploratory study lays the groundwork for future investigations into the causality of variations in exercise outcomes among individuals and different sexes, as well as the development of targeted interventions to promote physical activity and combat associated chronic diseases.