Abstract Clostridium difficile, a Gram-positive, anaerobic, sporogenic bacterium, is often seen in severely ill or elderly patients in hospitals or in long-term care facilities. Clostridium difficile infection (CDI), which is the most common cause of antibiotic-associated diarrhea in developed countries, is primarily caused by two homologous exotoxins, TcdA and TcdB. These toxins target and disrupt the colonic epithelium, leading to diarrhea and colitis through receptor mediated endocytosis. TcdA (~308 kDa) and TcdB (~270 kDa) contain four functional domains: an N-terminal glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a central transmembrane delivery and receptor-binding domain (DRBD), and a C-terminal combined repetitive oligopeptides (CROPs) domain. It is widely accepted that the toxins bind to cell surface receptors via the DRBD and the CROPs, and enter the cells through endocytosis. Acidification in the endosome triggers conformational changes in the toxins that prompt the DRBD to form a pore and deliver the GTD and the CPD across the endosomal membrane. In the cytosol, the CPD is activated by eukaryotic-specific inositol hexakisphosphate and subsequently undergoes autoproteolysis to release the GTD. The GTD then glucosylates small GTPases of the Rho family, including Rho, Rac, and Cdc42. Glucosylation of Rho proteins inhibits their functions, leading to alterations in the actin cytoskeleton, cell-rounding, and ultimately apoptotic cell death. Therefore the GTD is an ideal molecular target for therapeutic interventions, which directly targets the root cause of disease symptoms and cellular damage in CDI. While the relative roles of these two toxins in the pathogenesis of CDI are not completely understood, TcdB is considered to be more virulent than TcdA and more important for inducing the host inflammatory and innate immune responses. Therefore, we will focus on TcdB in this project, and the goal of this proposal is to understand the molecular mechanism by which TcdB covalently modifies its substrates, Rho family GTPases, by glucosylation. We propose two specific aims: (1) to understand the structural basis for recognition of Rho GTPases by the GTD, and (2) to understand the affinity and specificity requirements for the GTD–Rho recognition. We will use X-ray crystallography and structure- based mutagenesis to examine interactions between the GTD and Rho proteins at the molecular level, as well as to reveal the structural determinants of substrate specificity and vulnerabilities of the GTD. These findings will provide new insights into the function of the GTD and the pathogenicity of TcdB, which could guide the design of novel therapeutic reagents to treat CDI by inhibiting the activity of the GTD.