[Background] Genome editing with CRISPR-Cas9 technique may provide some new ways for gene knock-out, knock-in and gene editing in fungal pathogens. [Objective] To construct a system for gene editing in Colletotrichum gloeosporioides from Hevea brasiliensis. [Methods] Cas9 containing the nuclear localization signal peptide was expressed in Escherichia coli and purified. Potential cleavage sites of URA5 was analyzed and an SgRNA was transcribed in vitro. The Cas9 protein and SgRNA were assembled in vitro to form a ribonucleoprotein. The ribonucleoprotein was transformed into the protoplast of C. gloeosporioides and the transformants were screened. [Results] The Cas9 protein and SgRNA could form a stable ribonucleoprotein and this complex showed high DNA cleavage activity in vitro. The ribonucleoprotein was transformed into the protoplast of C. gloeosporioides and URA5 was successfully knocked out. [Conclusion] The system is convenient for gene editing in C. gloeosporioides of H. brasiliensis.