Stylo (Stylosanthes spp.) is a dominant leguminous forage, widely cultivated in tropical and subtropical areas. Anthracnose caused by Colletotrichum gloeosporioides is one of the most destructive diseases that limit the yield of stylo. Therefore, screening resistance varieties and improving the resistance of stylo are crucial strategies to control stylo anthracnose. In this study, the resistance evaluation of main variety RY2 and 39 stylo germplasms against C. gloeosporioides was performed, 12 stylo germplasms were rated as highly resistant (HR). Among them, 2001-84 was considered to be the most disease-resistant germplasm. Then, the stylo phenotype and the infection process of C. gloeosporioides revealed that the disease severity in resistant germplasm 2001-84 was significantly milder than susceptible germplasm RY2, and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione reductase (GR) and total antioxidant capacity (T-AOC) were higher than those in RY2 at different time post inoculation. Phosphoproteomics revealed that differentially phosphoproteins between 2001-84 and RY2 were significantly enriched in oxidoreductase activity at 96 hpi. An E3 ubiquitin ligase, SgATL31, were identified in both phosphoproteomics and plasma membrane proteomics, and overexpression of SgATL31 enhanced resistance to C. gloeosporioides in Arabidopsis. Additionally, SgATL31 promoted the accumulation of ROS in Arabidopsis leaves and stylo protoplasts under chitin treatment. Overall, this study identified the disease-resistant germplasm 2001-84 and elucidated that SgATL31 enhances resistance to anthracnose in Arabidopsis by inducing the accumulation of ROS. This study provided a reference for further exploring the functions of SgATL31 in C. gloeosporioides infection.