Abstract: Arginine is a functionally essential amino acid that plays a pivotal role in regulating reproductive endocrine functions and antioxidant status in teleosts, yet its specific requirements and regulatory mechanisms in largemouth bass (Micropterus salmoides) broodstock remain poorly understood. To evaluate the effects of graded dietary arginine (Arg) levels on the reproductive performance, health status, and underlying mechanisms of female M. salmoides broodstock, a 39-day feeding trial was conducted using mature females with an average body weight of (500.37 ± 23.42 g. Six experimental diets were formulated by supplementing 0% (A0), 0.5% (A0.5), 1.0% (A1), 1.5% (A1.5), 2.0% (A2), and 2.5% (A2.5) Arg, respectively. The results showed that dietary Arg levels significantly affected ovarian development. The gonadosomatic index (GSI) in the A1.5 diet was significantly higher than that in the A0 group, accompanied by a marked increase in the number of mature oocytes (P < 0.05). In addition, absolute fecundity (AF) and relative fecundity (RF) in the A1~A2.5 groups were significantly elevated compared with the A0 group (P < 0.05). Serum follicle-stimulating hormone (FSH) levels in the A1 and A1.5 groups were significantly increased relative to the A0 group (P < 0.05), while serum vitellogenin (VTG) and estradiol (E2) concentrations exhibited an upward trend across all Arg-supplemented groups. Dietary Arg supplementation significantly upregulated the relative expression of genes associated with ovarian development and steroidogenesis, including vitellogenin receptor gene (vtgr), follicle-stimulating hormone receptor gene (fshr), steroidogenic acute regulatory protein gene (star), and aromatase gene (cyp19a1). Moreover, Arg supplementation significantly reduced malondialdehyde (MDA) contents in both the liver and ovary, while enhancing the activities of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT). In conclusion, the promotion of ovarian development and reproductive performance in female M. salmoides broodstock by adequate Arg was achieved through the regulation of estrogen secretion, reproduction-related gene expression, and enhanced antioxidant capacity. Under the present experimental conditions, the optimal dietary Arg requirement for female M. salmoides broodstock, as determined by GSI and AF, was in the range of 3.84%-4.21% (7.87%-8.50% of dietary protein).