After labor expenses, the cost of fuel is the largest operating budget expense item for freight and passenger rail operations in the United States. Because fuel is such a costly component of operations, the railroad industry is constantly researching and testing new methods either to reduce the volume of fuel consumed or to switch to less-expensive fuels and sources of energy. An understanding of the factors that affect fuel consumption and their interactions is valuable for analyzing the feasibility of a given technology. Such knowledge allows for more intelligent extrapolation of simulation and laboratory results across a range of routes and in-service operating conditions. This research investigates the relative effects of infrastructure, equipment, and operating parameters on fuel efficiency for freight and passenger railroads on a mixed-use corridor. Partial factorial experiments investigate the effects of multiple factors on freight and passenger train fuel efficiency. Rail simulation software is used to run trial cases of single-track lines with heterogeneous traffic and to calculate energy-efficiency metrics. Results from the simulations are used to create two multivariate regression models for freight and passenger rail fuel efficiency. A sensitivity analysis identifies the relative effects of these factors on freight and passenger train fuel efficiency. By understanding the relative influence of various parameters on fuel efficiency, practitioners can focus data collection, modeling, and other fuel-saving efforts on the most significant factors.