Abstract: Withania somnifera (L.) Dunal (WS), or Ashwagandha, is used clinically against arthritis, anxiety and insomnia etc, however, its potential in aquafeeds is largely unexplored. We investigated the stress-alleviation and hepatoprotective mechanisms of dietary WS supplementation in adult female darkbarbel catfish. Fish were fed with WS dietary for 5 weeks, subjected to a physical stressor (saline injection), and sampled. Brain were processed for RNA-seq to identify differentially expressed genes (DEGs) linked to stress; serum were assayed for stress- and immune-related biochemical markers; livers were examined for lipid content (Oil Red O staining) and proliferation (PCNA immunostaining). Network pharmacology were employed to construct a "compound-target" network: The bioactive compounds of WS were retrieved from ChEBI and PCIDB, probable targets predicted via Swiss Target Prediction, and the “WS-ingredient-target” regulatory network was constructed using Cytoscape; key ligand-protein interactions were validated by molecular docking. Findings were replicated in juvenile fish and in SG3 cells challenged with Withanolide A. Physical stress evoked 1 451 DEGs in control brains, whereas WS-supplemented reduced this to 397, indicating marked mitigation of the oxidative- and immune-stress response. Network pharmacology and molecular docking predicted that 13 bioactive compounds in WS, especially Withanolide A and Withanolide J, can interact with targets such as MAPK8, CRHR1, NR3C2, and IARS1, being associated with stress and liver function regulation respectively. Moreover, it was proved that Withanolide A could significantly modulate stress response in juveniles and enhanced proliferation potency of SG3 cells. Intriguingly, in theadult female darkbarbel catfish, dietary WS lowered serum cortisol, glucose, ROS, TNF-α, and IL-6 ( P < 0.05), up-regulated hepatocyte PCNA expression, and allievated stress-induced liver injury. These findings provide novel insights and vital basis for elucidating the mechanisms whereby herbs, including WS act in stress-relief and hepatoprotection in darkbarbel catfish. Andprovide a framework for advancing traditional Chinese medicine (TCM) applications in aquaculture.