This life cycle assessment evaluates and quantifies the environmental impacts of the production of hydrogen and transportation fuels from the fast pyrolysis and upgrading of corn stover. Input data for this analysis come from Aspen Plus modeling, a GREET (Greenhouse Gases, Regulated Emissions, and Energy Use in Transportation) model database and a US Life Cycle Inventory Database. SimaPro 7.3 software is employed to estimate the environmental impacts. The results indicate that the net fossil energy input is 0.25 MJ and 0.23 MJ per km traveled for a light-duty vehicle fueled by gasoline and diesel fuel, respectively. Bio-oil production requires the largest fossil energy input. The net global warming potential (GWP) is 0.037 kg CO2eq and 0.015 kg CO2eq per km traveled for a vehicle fueled by gasoline and diesel fuel, respectively. Vehicle operations contribute up to 33% of the total positive GWP, which is the largest greenhouse gas footprint of all the unit processes. The net GWPs in this study are 88% and 94% lower than for petroleum-based gasoline and diesel fuel (2005 baseline), respectively. Biomass transportation has the largest impact on ozone depletion among all of the unit processes. Sensitivity analysis shows that fuel economy, transportation fuel yield, bio-oil yield, and electricity consumption are the key factors that influence greenhouse gas emissions.