Abstract: Oreochromis niloticus is one of the most important freshwater farmed fish species globally. Enhancing its muscle quality, particularly the content of n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs) and its overall texture, is beneficial in meeting consumer health demands and increasing product value-added. Feed is a key factor regulating the growth and muscle nutrient composition of farmed fish. The saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) it contains play vital roles in fish growth and the n-3 LC-PUFA sparing effect. Therefore, to investigate the optimal dietary SFA/MUFA ratio for O. niloticus, we formulated four isoproteic (30% crude protein) and isolipidic (7% crude lipid) experimental diets. These diets (T1-T4) had SFA/MUFA ratios of 0.31, 0.52, 0.97, and 1.52, respectively, and were formulated using soybean oil, rapeseed oil, and beef tallow as lipid sources. Juvenile O. niloticus (initial body weight: 5.0 g) were fed these diets for 9 weeks. No significant differences were observed in growth performance, feed conversion ratio, whole-body proximate composition, serum LDL-C, MDA levels, or T-AOC capacity among the groups. However, serum HDL-C, T-CHO, and TG levels increased with higher dietary SFA/MUFA ratios. Regarding muscle quality, the hardness, chewiness, and springiness of muscle in the T1 and T2 groups were significantly improved compared to the T3 and T4 groups. In terms of lipid metabolism, the n-3 LC-PUFA content in the liver and muscle was highest in the T1 group, although the mRNA expression levels of key LC-PUFA biosynthesis-related genes (fads2 and elovl5) in the liver showed no significant differences across groups. Additionally, the cytoplasmic NAD⁺/NADH ratio significantly decreased with increasing dietary SFA/MUFA ratios. Notably, the expression levels of fatty acid catabolism-related genes (aco-x1 and cpt-1a) and synthesis-related genes (fas and acc) in muscle reached their maximum in the T1 group. In conclusion, a dietary SFA/MUFA ratio of 0.31 enhances muscle fatty acid nutrition and texture quality in O. niloticus. The improved tissue n-3 LC-PUFA levels are likely due to the "n-3 LC-PUFA sparing effect" facilitated by a low SFA/MUFA ratio in the diet rather than enhanced endogenous LC-PUFA biosynthesis. These findings provide novel insights into optimizing dietary fatty acid strategies for cultivating high-quality O. niloticus.