Technical Abstract: Background: Biofuels offer a viable alternative to petroleum-based fuel. However, current methods are not sufficient and technology to use lignocellulosic biomass as a fermentation substrate faces several challenges. One challenge is the need of a robust fermentative microorganism that can tolerate the inhibitors present during lignocellulosic fermentation. These inhibitors include the furan aldehyde, furfural, which is released as a byproduct of xylose during weak acid pretreatment of lignocellulose. To survive in the presence of furfural yeast need to not only reduce furfural to the less toxic furfuryl alcohol, but also protect themselves and repair any damage caused by furfural. Since furfural tolerance in yeast requires a functional pentose phosphate pathway (PPP), and the PPP is associated with reactive oxygen species (ROS) tolerance, we investigated whether or not furfural induces ROS and ROS related cellular damage. Results: We have demonstrated that furfural induces the accumulation of ROS. In addition, furfural was shown to cause cellular damage that is consistent with ROS accumulation in cells. This includes damage to mitochondria and vacuole membranes, the actin cytoskeleton, and nuclear chromatin, but not damage to the external structure of the cell. At non-lethal concentrations of furfural (25 mM), the damage is less severe compared to lethal concentrations (50 mM). The less severe damage may be a factor in the cells ability to survive after the furfural has been detoxified. Conclusion: These data suggest that during the furfural-induced lag time, yeast not only detoxify furfural into furfuryl alcohol, but also protect themselves from the cellular effects of furfural and repair any damage caused by furfural. Understanding the cellular effects of furfural will aid in engineering robust yeast strains capable of surviving the stresses associated with lignocellulosic fermentation.