Abstract: Under intensive aquaculture systems, largemouth bass (Micropterus salmoides) is highly susceptible to nutritional metabolic disorders, which severely compromise growth performance and muscle quality. Oxaloacetic acid (OAA), a critical intermediate in the tricarboxylic acid (TCA) cycle, plays a pivotal regulatory role in regulating energy metabolism homeostasis and holds potential as a functional feed additive. However, its regulatory effects on aquaculture species remain unclear. This study investigated the effects of dietary oxaloacetic acid (OAA) supplementation on growth performance, glucose-lipid metabolism, and muscle fiber development in largemouth bass. Juvenile largemouth bass initial weight: (8.79 ± 0.04) g were fed four isonitrogenous and isolipidic diets: control (C), 0.5% OAA (LA), 1% OAA (MA), and 2% OAA (HA), with three replicates per group, for 8 weeks. Compared to the control group, OAA supplementation significantly decreased weight gain rate and whole-body total lipid content, while increasing feeding rate. No significant differences were observed in feed conversion ratio, carcass ratio, or whole-body total protein content. Serum glucose, pyruvate, lactate, insulin, and muscle glycogen levels showed no significant differences among groups, while hepatic glycogen in the LA group significantly increased compared to the control group. Hepatic citrate content decreased progressively with increasing OAA inclusion level, whereas succinate dehydrogenase (SDH) activity increased significantly. The HA group exhibited a significant decrease in the NADH/NAD⁺ ratio in muscle tissue, accompanied by a significant increase in liver NADH content and muscle NAD⁺ content. Hepatic total lipid content and results of Oil Red O staining revealed reduced lipid droplet accumulation in the HA group, accompanied by decreased total lipid and triglyceride (TG) levels in muscle. Furthermore, OAA supplementation downregulated lipid synthesis genes (fas, pparγ, srebp2, lipin1) and upregulated lipid catabolism genes (atgl, lpla, pparα, cpt1a) in liver and muscle. Interestingly, OAA significantly increased the proportion of small-diameter muscle fibers (20–50 μm) while reducing that of large-diameter fibers (110–140 μm). The expression of myogenic regulatory factors (myog, myod) was significantly upregulated by OAA. Dietary OAA supplementation reduced lipid deposition, enhanced TCA cycle activity and promoted muscle fiber proliferation and differentiation in largemouth bass. This study revealed the important role of OAA in regulating glucose and lipid metabolism and myofiber growth and development in fish, and also provided a new choice of feed additive for the aquaculture industry.