Stylosanthes guianensis is a dominant pasture legume in tropical and subtropical regions. Anthracnose, a fungal disease caused by Colletotrichum gloeosporioides (C. gloeosporioides), significantly limits the production of S. guianensis. Phenylalanine ammonia-lyase (PAL), the rate-limiting enzyme in the phenylpropanoid pathway, plays a vital role in regulating plant resistance to pathogens. However, its function in the stylo’s resistance against anthracnose requires further exploration. In this study, a core collection (28 accessions) representing 237 S. guianensis accessions was established based on phenotypic data and SSR (Simple Sequence Repeat) molecular marker data. The PAL activity in response to C. gloeosporioides was correlated with the resistance phenotypes in the core collection. Five members of the SgPAL gene family were identified from previous transcriptomic data. Among these, SgPAL1 and SgPAL2 showed a significant positive correlation with the PAL activity across the core collection after inoculation. Biochemical analyses, subcellular localization study, and expression pattern analyses demonstrated that SgPAL1 and SgPAL2 displayed PAL activity, localized in the cytoplasm, and were induced by C. gloeosporioides inoculation. Furthermore, overexpression (OE) of SgPAL1 and SgPAL2 in Arabidopsis enhanced resistance against C. gloeosporioides. The transcriptomic analyses of SgPAL1 and SgPAL2 OE lines revealed the up-regulation of the key genes regulating the lignin synthesis. Consistently, the total lignin content and the ratio of G to S monomers were also higher in SgPAL1 and SgPAL2 OE lines than in wild type plant after inoculation. These results imply that SgPAL1 and SgPAL2 may enhance plant resistance against C. gloeosporioides by increasing lignin content and the G/S ratio. In conclusion, this study provides an effective core germplasm collection for advancing the genetic breeding of S. guianensis, and characterize SgPAL1 and SgPAL2 as promising targets for improving crop resistance against anthracnose.