Clostridioides difficile is a spore-forming, Gram-positive, obligate anaerobe majorly implicated in nosocomial infections. It causes antibiotic-induced nosocomial diarrhoeas and related healthcare infections in most parts of the world. Dissemination of C. difficile infections occurs mainly via surfaces contaminated with spores especially in clinical settings and disease control is significantly hindered by spore formation. Spores are highly infectious, and resistant to heat, alcohol, and standard disinfectants. Transglutaminases are produced during sporulation in Streptomyces mobaraense and Bacillus subtilis and promote survival and virulence by modulating cellular protein crosslinking which increases resistance of cellular structures. Transglutaminases are enzymes in microbial and mammalian organisms that irreversibly crosslink proteins by forming covalent epsilon (gamma-glutamyl) lysine bonds that are proteolysis-resistant. The detection of novel important genes not explored previously by antibiotics can facilitate the discovery of alternative efficient antimicrobials to surmount existing resistance associated with disease control and treatment. The aim of this study was to identify novel transglutaminase-like genes associated with sporulation in C. difficile 630. We detected transglutaminase activity in sporulating cells and spores of CD 630. Also, transglutaminase gene expression was detected during sporulation. Three transglutaminase-like genes were identified in CD 630 and successfully cloned in various vectors and expressed in E. coli. No transglutaminase activity was detected in culture supernatants and in purified recombinant proteins. However, transglutaminase-like proteins were purified from sporulating C. difficile 630 cells by a two-step strategy to attain a specific activity and purification fold of 4 and 57 respectively. The purified proteins were characterized and found to be calcium-independent like most microbial transglutaminases, significantly inhibited by PMSF, and only inhibited to a small extent by thiol group inhibitors. Mass spectrometry analysis of the purified proteins suggests that they are mainly involved in metabolism in CD 630. We conclude that CD630 produces transglutaminase-like proteins during sporulation whose role necessitates functional analysis involving gene knockouts and the associated phenotypes of spores.