Contamination of fresh produce with Listeria monocytogenes can occur throughout the supply chain, including at retail, where Listeria spp., including L. monocytogenes, may be introduced and spread via various routes. However, limited tools are available for retailers to assess practices that can enhance control of Listeria transmission to fresh produce. Therefore, we developed an agent-based model that can simulate Listeria transmission in retail produce sections to optimize environmental sampling programs and evaluate control strategies. A single retail store was used as a model environment, in which various routes of Listeria introduction into and transmission between environmental surfaces were modeled. Model prediction (i.e., Listeria prevalence) was validated using a published longitudinal study for all surfaces that were included in both the model and the validation data. Sensitivity analysis using the Partial Rank Correlation Coefficient showed that (i) initial Listeria concentration from incoming produce, (ii) transfer coefficient from produce to employee's hands, and (iii) transfer coefficient from consumer to produce were the top three parameters that were significantly (p < 0.0018) associated with the mean Listeria prevalence across all agents, suggesting that the accuracy of these parameters are important for prediction of overall Listeria prevalence at retail. Cluster analysis grouped agents with similar contamination patterns into six unique clusters; this information can be used to optimize the sampling plans for retail environments. Scenario analysis suggested that (i) more stringent supplier control as well as (ii) practices reducing Listeria transmission via consumer's hands may have the largest impact on reducing finished product contamination. Overall, we show that an agent-based model can serve as a foundational tool to help with decision-making on Listeria control strategies at retail.