Blackground/Objectives: Tributyrin (TB), a stable derivative of butyric acid, has been widely used in animal feeds for its health-promoting effects. This study evaluated the efficacy of dietary TB serving as a functional feed additive in enhancing intestinal health of mandarin fish (Siniperca chuatsi), with a focus on elucidating the associated molecular mechanisms. Methods: A total of 300 juvenile mandarin fish (200.0 ± 5.2 g) were randomly assigned to one of three dietary groups: a control group (0 mg/kg TB), a low-dose TB group (TB1, 500 mg/kg), or a high-dose TB group (TB2, 1000 mg/kg). The one-month feeding trial was conducted under strictly controlled conditions, with water quality maintained within optimal range. Fish were fed their respective diets twice daily to apparent satiety. Results: Results showed that TB supplementation significantly increased villus height in the mid- and hindgut, with the TB2 group showing the most pronounced improvement. Furthermore, transcriptome analysis revealed that TB altered the expression of genes involved in energy metabolism, fatty acid oxidation, and steroid biosynthesis pathways. Notably, TB supplementation up-regulated key genes such as gls2b (energy metabolism) and cpt1b (fatty acid oxidation), and modulated especially modulated steroid biosynthesis through genes sqlea and dhcr24h. Co-expression network analysis further identified hub genes associated with energy metabolism (etfb), immune regulation (il20ra, foxp1b), and cell cycle regulation (cdc20, ccnb1). Conclusions: These findings elucidate the mechanism of action of TB as a functional feed additive, providing a theoretical foundation for its application in aquaculture to enhance intestinal health.