All cell membranes are packed with proteins. The ability to investigate the regulatory mechanisms of protein channels in experimental conditions mimicking their congested native environment is crucial for understanding the environmental physicochemical cues that may fundamentally contribute to their functionality in natural membranes. Here we report on investigations of the voltage-induced gating of lysenin channels in congested conditions experimentally achieved by increasing the number of channels inserted into planar lipid membranes. Typical electrophysiology measurements reveal congestion-induced changes to the voltage-induced gating, manifested as a significant reduction of the response to external voltage stimuli. Furthermore, we demonstrate a similar diminished voltage sensitivity for smaller populations of channels by reducing the amount of sphingomyelin in the membrane. Given lysenin’s preference for targeting lipid rafts, this result indicates the potential role of the heterogeneous organization of the membrane in modulating channel functionality. Our work indicates that local congestion within membranes may alter the energy landscape and the kinetics of conformational changes of lysenin channels in response to voltage stimuli. This level of understanding may be extended to better characterize the role of the specific membrane environment in modulating the biological functionality of protein channels in health and disease.