Wildlife-vehicle collisions (WVC) can result in property damage, injury and death to drivers. WVC can similarly cause wildlife injury and mortality, as well as genetic fragmentation and other population impacts. State departments of transportation seeking to evaluate the need to reduce wildlife-vehicle collisions use evidence of WVC and other data to inform decisions about WVC mitigation. Mitigation infrastructure, such as wildlife crossings with exclusion fencing, are effective measures for reducing WVC. Yet, states generally lack consistent mechanisms for evaluating the economic and societal benefits of reducing WVC relative to the costs of mitigation. We developed a web-based tool (https://www.wildlifecrossingcalculator.org) to assist decision-making related to WVC mitigation, based on economic benefits and costs associated with WVC and mitigation activities. The tool provides a portal for states and their partners to estimate benefits and costs and total value of proposed projects for WVC mitigation. The tool was based on the premise that “benefits” are the sum of avoidable costs (e.g., reduced crashes involving wildlife) and “costs” are the sum of response costs, such as constructed mitigation. We collaborated with transportation and wildlife staff from 5 US states (AZ, ID, MT, OR, WY) to elucidate goals for such a decision-support tool, develop wildlife values, estimate crash costs, and calculate costs for different types of mitigation. The workflow for the tool begins with the registered user defining a study area (e.g., state, project area) and selecting either default wildlife and crash costs for their state or entering new values. The user can also use default mitigation costs or enter their own. The tool estimates the total costs (wildlife cost + crash cost) for individual WVC events, which can then be summed to a roadway segment (e.g., 1 mile section). The user can then compare the benefit of avoiding the costs of WVC with potential costs of mitigation. The Wildlife Crossing Calculator provides flexibility and transparency in comparing the fiscal benefits and costs of potential mitigation investments to support how states prioritize and incorporate WVC reduction projects into transportation planning.