Guayule (Parthenium argentatum) is a perennial shrub that can be cultivated in the Southwestern US. It produces natural rubber that could be a viable substitute for Hevea natural rubber and synthetic rubbers currently used in tires. Drivers for producing domestic guayule rubber include fluctuations in price and availability of imported Hevea rubber. A tire was manufactured in 2017 where guayule rubber was substituted for all of the Hevea and synthetic rubber in the conventional tire. Life cycle assessment (LCA) from cradle to grave was used to evaluate the environmental and energy sustainability of the guayule tire, and these metrics were benchmarked against those of the conventional tire (CT). Functional units of 1 kg natural rubber for agricultural processes, as well as 1 tire for the cradle-to-grave study were considered. Life cycle inventory (LCI) data were collected directly from primary sources, including guayule field experiments, a rubber-processing company, and a major tire manufacturer. Scenario analysis was used to evaluate alternative processes, such as irrigation options in guayule cultivation, processing scale in rubber extraction, and selection of allocation methodology in LCAs. Model uncertainty was characterized using Monte Carlo analysis. The LC energy consumption of the guayule tire (GT) was 13.7 GJ/tire (including co-product credits, excluding C sequestration during agriculture), compared to 16.4 GJ/tire for the CT. The GT had 6–30% lower emissions than CT in ten different environmental impact categories. Bagasse co-product in energy applications showed benefits of reducing energy consumption by 10% and decreasing environmental impacts by up to 11%. GT’s use-phase resulted in the highest energy consumption (95%) and environmental impacts ranging between 81 and 99%. Variables in use phase, i.e., rolling resistance coefficient, vehicle efficiency, and tire lifetime, and those in guayule cultivation i.e., rubber and biomass yields, were key model parameters. The effect of excluded but potentially important model parameters, i.e., guayule carbon sequestration and resin co-product were tested via sensitivity analyses. Based on these results that factored in the lower rolling resistance coefficient of a guayule tire—a significant element that improves the fuel economy of an automobile—the guayule rubber tire shows promise in its ability to replace current conventional tires. The first commercially manufactured guayule rubber passenger tire will most likely substitute components, which are either partially or fully guayule, instead of guayule replacing 100% of the existing rubbers as shown in this study.