Glomerella leaf spot (GLS) is a severe infectious dis- ease of apple whose infective area is growing gradually and thus poses a huge economic threat to the world. Different species of Colletotrichum including Colleto- trichum gloeosporioides are responsible for GLS. For efficient GLS control, it is important to understand the mechanism by which the cruciferous crops and C. gloeosporioides interact. Arginine is among one of the several types of amino acids, which plays crucial role in biochemical and physiological functions of fungi. The arginine biosynthesis pathway involved in virulence among plant pathogenic fungi is poorly understood. In this study, CgCPS1 gene encoding carbamoyl phos- phate synthase involved in arginine biosynthesis has been identified and inactivated experimentally. To as- sess the effects of CgCPS1, we knocked out CgCPS1 in C. gloeosporioides and evaluated its effects on virulence and stress tolerance. The results showed that deletion of CgCPS1 resulted in loss of pathogenicity. The Δcgcps1 mutants showed slow growth rate, defects in appres- sorium formation and failed to develop lesions on apple leaves and fruits leading to loss of virulence while complementation strain (CgCPS1-C) fully restored its pathogenicity. Furthermore, mutant strains showed extreme sensitivity to high osmotic stress displaying that CgCPS1 plays a vital role in stress response. These findings suggest that CgCPS1 is major factor that me- diates pathogenicity in C. gloeosporioides by encoding carbamoyl phosphate that is involved in arginine bio- synthesis and conferring virulence in C. gloeosporioides.