This life cycle assessment of Jatropha biodiesel production and use evaluates the net greenhouse gas (GHG) emission (not considering land-use change), net energy value (NEV), and net petroleum consumption impacts of substituting Jatropha biodiesel for conventional petroleum diesel in India. Several blends of biodiesel with petroleum diesel are evaluated for the rail freight, rail passenger, road freight, and road-passenger transport sectors that currently rely heavily on petroleum diesel. For the base case, Jatropha cultivation, processing, and use conditions that were analyzed, the use of B20 results in a net reduction in GHG emissions and petroleum consumption of 14% and 17%, respectively, and a NEV increase of 58% compared with the use of 100% petroleum diesel. While the road-passenger transport sector provides the greatest sustainability benefits per 1000 gross tonne kilometers, the road freight sector eventually provides the greatest absolute benefits owing to substantially higher projected utilization by year 2020. Nevertheless, introduction of biodiesel to the rail sector might present the fewest logistic and capital expenditure challenges in the near term. Sensitivity analyses confirmed that the sustainability benefits are maintained under multiple plausible cultivation, processing, and distribution scenarios. However, the sustainability of any individual Jatropha plantation will depend on site-specific conditions.