Anthocyanins are important secondary metabolites in fruits, and anthocyanin accumulation in the flesh of peach exhibits a spatial pattern, but the relevant mechanism is still unknown. In this study, the yellow-fleshed peach, cv. ‘Jinxiu’, with anthocyanin accumulation in the mesocarp around the stone was used as the experimental material. Red flesh (RF) and yellow flesh (YF) were sampled separately for flavonoid metabolite (mainly anthocyanins), plant hormone, and transcriptome analyses. The results showed that the red coloration in the mesocarp was due to the accumulation of cyanidin-3-O-glucoside, with an up-regulation of anthocyanin biosynthetic genes (F3H,F3′H,DFR, andANS), transportation geneGST, and regulatory genes (MYB10.1andbHLH3). ElevenERFs, nineWRKYs, and eightNACswere also defined as the candidate regulators of anthocyanin biosynthesis in peach via RNA-seq. Auxin, cytokinin, abscisic acid (ABA), salicylic acid (SA), and 1-aminocyclopropane-1-carboxylic acid (ACC, ethylene precursor) were enriched in the peach flesh, with auxin, cytokinin, ACC, and SA being highly accumulated in the RF, but ABA was mainly distributed in the YF. The activators and repressors in the auxin and cytokinin signaling transduction pathways were mostly up-regulated and down-regulated, respectively. Our results provide new insights into the regulation of spatial accumulation pattern of anthocyanins in peach flesh.