This study aimed to investigate the role of the protein phosphatase 2A regulatory subunit a (PP2Aa) in limb regeneration in Periplaneta americana. Bioinformatics analyses were conducted to assess the phylogenetic relationship and amino acid sequence conservation of the PP2Aa protein. Quantitative real-time PCR (RT-PCR) was used to measure PP2Aa expression at various time points following limb amputation. The results revealed that PP2Aa expression upregulated between 3 and 120 hours post-amputation, with a subsequent decline after 240 hours. These findings suggest that PP2Aa was involved in blastema formation and morphogenesis during limb regeneration in P. americana. Furthermore, the injection of PP2Aa dsRNA to inhibit its expression significantly impaired limb regeneration of P. americana, highlighting the critical role of PP2Aa in this process. Further studies using EdU and DAPI staining confirmed that the disruption of PP2Aa expression inhibited cell proliferation, preventing the formation of a visible regenerated limb at the amputation site. These results suggested that PP2Aa played a crucial role in promoting limb regeneration by regulating cell proliferation. As a widely-distributed and evolutionarily conserved serine/threonine phosphatase, PP2A may serve as a key regulator in this process. These findings lay the groundwork for further exploration of the limited regenerative capacity of higher mammals.