Fruit color influences fruit quality and commodity value. Most longan (Dimocarpus longan Lour.) varieties have a yellowish-brown or grayish-yellow pericarp, and the discovery of red pericarp (RP) longan expanded the color varieties of longan fruit. Our previous research showed that the red pericarp of RP fruit was mainly caused by anthocyanin accumulation; however, its underlying regulatory mechanism remains unknown. Herein, DlMYB113, an R2R3-MYB transcription factor, was discovered by examining differentially expressed genes in two longan cultivars. Dimocarpus longan MYB (v-myb avian myeloblastosis viral) 113 (DlMYB113) expression was significantly higher in the pericarp and leaves of RP longan than in ‘Shixia’ longan. Sequence alignment analysis revealed two amino acid substitutions in the R3 domain between DlMYB113rp in RP longan and DlMYB113sx in ‘Shixia’ longan. Transient expression of DlMYB113rp significantly increased anthocyanin accumulation in tobacco leaves, whereas DlMYB113sx had negligible effect. Meanwhile, DlMYB113 overexpression promotes anthocyanin accumulation in Arabidopsis and longan calli. Site-directed mutation detection revealed divergence in DlMYB113 function when the R3 repeat 197-position base T was replaced with G, and the 317- and 318-position AT bases were replaced with GA. Our findings indicate that DlMYB113 can regulate anthocyanin production in RP longan, and three mutations in its nucleic acid sequence lead to anthocyanin accumulation, thereby developing molecular markers associated with the anthocyanin accumulation trait in RP longan. This study will facilitate early screening of longan hybrids with desirable fruit color and be significant for breeding new characteristic varieties.