Glycogen serves as a crucial indicator for assessing the quality of oysters, and glycogen debranching enzyme (AGL) plays a pivotal role in glycogenolysis. The Jinjiang oyster (Crassostrea ariakensis), widely distributed along China’s coast, is highly sought after in the high-end market. However, the molecular mechanism of glycogen metabolism in the Jinjiang oyster has been inadequately investigated. This study aimed to explore the AGL gene expression during the proliferative stage by administering dsRNA, and subsequently to investigate the relationship between the mRNA expression and glycogen content, thereby elucidating the pivotal role of this gene in glycogen metabolism. First, bioinformatics tools were employed to analyze the AGL gene sequence. Subsequently, dsRNA expression vectors were constructed for inducing the dsRNA production by the HT115 (DE3) bacteria, which were then co-cultured with unicellular algae as attachment hosts. Finally, the bacterial-algal mixture was fed daily to the Jinjiang oyster for RNA interference (RNAi). Furthermore, on days 15 and 30 of RNAi treatment, gonadal tissues were collected for subsequent determination of glycogen content and gene expression analysis. In the control group, gonadal development was observed using tissue sections. The software SPSS 26 was employed to analyze the correlation between AGL gene expression and glycogen content in gonads as well as to determine differences in data. According to the findings, the coding region of the AGL gene had a sequence length of 4719 bp and encoded 1572 amino acids. It contained four structural domains and exhibited a predicted protein molecular weight of 178.23 kDa, a theoretical isoelectric point of 6.21, and an AGL sequence similarity with other selected species ranging from 49.59% to 97.26%. Phylogenetic analysis revealed that the C. ariakensis AGL gene was genetically most closely related to mollusks. Throughout the experimental period, gonads in the control group showed progressive development but remained in the proliferative stage. Following interference for 15 and 30 days, expression levels of the AGL gene were significantly lower (P＜0.05) while glycogen content was significantly higher (P＜0.05) in comparison to those in the control group. In addition, after RNAi for 30 days compared to 15 days, there was a significant increase (P＜0.05) in AGL gene expression accompanied by a significant decrease (P＜0.05) in glycogen content. Importantly, it was observed that there existed a highly significant strong negative correlation (P＜0.05) between AGL gene expression and glycogen content within C. ariakensis gonads. In this study, we successfully employed the RNAi technology to downregulate the gene expression of AGL in the Jinjiang oyster and observed corresponding alterations in glycogen content, thereby confirming the critical role of the AGL gene in glycogenolysis. By manipulating the gene expression to validate its function, it is anticipated that the artificial regulation of glycogen content and other quality traits can be achieved, ultimately enhancing oyster quality and increasing market value. The present study contributes to the comprehensive understanding of the AGL gene in oyster glycogen metabolism, thereby offering valuable insights for the exploration of novel technologies aimed at regulating glycogen content.