Persistent hot and humid tropical climate induces heat stress (HS), negatively impacting gut integrity with inflammation and tight junction degradation in broilers. As integrating operations of the digestive tract and hypothalamus in avian are regulated by feed intake, the effect of HS may be eased by feed supplementation. Herein, the effect of isoquinoline alkaloids (IQ) supplementation from Macleaya cordata on integrating gut-brain axis and gut integrity was assessed. 720 male broilers (Ross 308) were treated with IQ at null (control), 60 (IQ60), and 100 mg/kg (IQ100). They were reared in open production house between April and May in Southern Thailand with 33.0–38.0°C (average 35.0°C), 75–80% relative humidity (RH), and 160–200 heat stress index (HI). From day 1 to 42, IQ supplementation positively affected growth performance where body weight (BW), body weight gain (BWG), and feed intake (FI) were significantly higher in birds fed IQs than in the control group (P<0.05), and feed conversion ratio (FCR) was significantly lower in the IQ group (P<0.05). IQ improved gut integrity by reducing inflammation with significant down-regulation of nitric oxide synthase (iNOS), cytokines interferon gamma (IFN-γ), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), and nuclear factor κB (NF-κB) in the ileum (P<0.05). It strengthened tight junction by upregulates occludin (OCLD), claudin-1 (CLDN1), tight junction proteins-1 (TJP-1) and mucin-2 (MUC-2) in the jejunum (P<0.05). As a result, serum fluorescein isothiocyanate-dextran (FITC-d) and corticosterone levels were significantly lower (P<0.05). In terms of the gut-brain axis, IQ supplementation reduces ghrelin and cholecystokinin (CCK) mRNA expression in the jejunum and hypothalamus (P<0.05), which downregulated the expression of pro-opiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), corticotropin-releasing hormone (CRH), and melanocortin four receptor (MC4R). It was the first evidence that IQ influences on modulating the gut-brain axis to suppress anorexigenic regulation in broilers with HS exposure. © 2023