Acetylcholine (ACh), a potent neuromodulator in the central nervous system, plays a pivotal role in visual processing, attention, and cognitive flexibility. Most of the studies have been using standard visual stimuli, but the advance of virtual reality (VR) tools is promising for better elucidating the dynamics of this neuromodulator in visually guided behaviors. The present study aims to explore the spatiotemporal activation of cortical cholinergic fibers during the presentation of a VR dynamic environment compared to standard visual stimuli using mesoscopic ACh imaging in awake mice.Cholinergic activity in AAV-delivered gACh-3.0 awake mice (n=6) was compared to calcium activity of Thy1-GCaMP6s mice (n=6) using wide-field mesoscopic imaging. Two gaming monitors displayed either a sinusoidal grating, Random Dot Kinematogram, or a Unity-generated VR sequence in motion. ACh release and calcium cortical responses (ΔF/F, %) were measured in 10 various areas, including V1 in the head-fixed mouse during standard and VR stimuli.During grating or RDK stimulation, most visual areas exhibited activation up to 60% in V1 for ACh release and 100% for gCAMP6s in a contrast-dependent manner. VR triggered significant ACh release across visual, motor, sensorimotor, and associative areas at different time points.ACh release was evoked throughout the cortical mantle by the dynamic VR sequence, whereas it was more limited to the visual areas in response to standard visual stimuli. This observation implies the important role of ACh in visual perception.