Abstract The cortical distribution and functional role of cholecystokinin (CCK) are largely unknown. Here, a CCK receptor antagonist challenge paradigm was developed to assess functional connectivity and neuronal responses. Structural–functional magnetic resonance imaging and calcium imaging were undertaken in environmental enrichment (EE) and standard environment (SE) groups (naïve adult male mice, n = 59, C57BL/B6J, P = 60). Functional connectivity network-based statistics and pseudo-demarcation Voronoi tessellations to cluster calcium signals were used to derive region of interest metrics based on calcium transients, firing rate, and location. The CCK challenge elicited robust changes to structural–functional networks, decreased neuronal calcium transients, and max firing rate (5 s) of dorsal hippocampus in SE mice. However, the functional changes were not observed in EE mice, while the decreased neuronal calcium transients and max firing rate (5 s) were similar to SE mice. Decreased gray matter alterations were observed in multiple brain regions in the SE group due to CCK challenge, while no effect was observed in the EE group. The networks most affected by CCK challenge in SE included within isocortex, isocortex to olfactory, isocortex to striatum, olfactory to midbrain, and olfactory to thalamus. The EE group did not experience network changes in functional connectivity due to CCK challenge. Interestingly, calcium imaging revealed a significant decrease in transients and max firing rate (5 s) in the dorsal CA1 hippocampus subregion after CCK challenge in EE. Overall, CCK receptor antagonists affected brain-wide structural–functional connectivity within the isocortex, in addition to eliciting decreased neuronal calcium transients and max firing rate (5 s) in CA1 of the hippocampus. Future studies should investigate the CCK functional networks and how these processes affect isocortex modulation. Significance Statement Cholecystokinin is a neuropeptide predominately found in the gastrointestinal system. Albeit abundantly expressed in neurons, the role and distribution of cholecystokinin are largely unknown. Here, we demonstrate cholecystokinin affects brain-wide structural–functional networks within the isocortex. In the hippocampus, the cholecystokinin receptor antagonist challenge decreases neuronal calcium transients and max firing rate (5 s) in CA1. We further demonstrate that mice in environmental enrichment do not experience functional network changes to the CCK receptor antagonist challenge. Environmental enrichment may afford protection to the alterations observed in control mice due to CCK. Our results suggest that cholecystokinin is distributed throughout the brain, interacts in the isocortex, and demonstrates an unexpected functional network stability for enriched mice.