Hydrogen peroxide, methylation and alternative splicing (AS) all play important roles in fruit development, but the regulatory relationship between them in fruit development is unclear. In this study, AS events occurring at different developmental stages of the H2O2 treated and control berries of Kyoho from 35 to 65 days post anthesis were analyzed based on RNA-seq data. The results showed that alternative 3′ splice site (A3) was the most type and mutually exclusive exons (MX) was the least type revealed in both the treatment and control. There was stage-specific regulation of AS during grape berries development and the events of AS were more abundant at the early stages of berry development, and 11,670 was identified as the conserved events during berry development. There were 657 AS events which showed significant differences between the treatment and control, and functional annotation demonstrated that 10 of these genes were associated with methylation modifications. The significant differences in the extent of AS of these methylation related genes in the treatment and control suggest that AS, methylation modification and H2O2 synergistically regulate the grape berries ripening process.