High-fat diet (HFD)-induced obesity is a global health concern associated with gastrointestinal disorders. While mammalian models have elucidated the effects of a HFD on intestinal structure and function, its impact on zebrafish, a crucial model for studying diet-induced obesity and gastrointestinal dysfunction, remains inadequately characterized. This study investigated the influence of a HFD on zebrafish intestinal morphology, tight junction (TJ) protein expression, and inflammatory markers. Zebrafish fed a control diet or HFD with 40% or 60% fat exhibited significant alterations in intestinal morphology, with increased villi number but reduced villi width and length, suggesting compensatory responses to dietary stress. TJ protein expression (Claudin 2, Claudin 3, and Claudin 10) showed complex changes, particularly in the HFD60 juvenile group, indicating a multifaceted response in barrier integrity. Pro-inflammatory cytokine IL-6 and TNF-α levels were lower in both the juvenile and adult HFD60 groups than in the HFD40 and control groups, while elevated anti-inflammatory IL-10 levels in HFD60 adult zebrafish suggested activation of compensatory mechanisms. These findings highlight zebrafish as a valuable model for studying the effects of HFD on intestinal health and provide insights into the relationship between dietary fat, gut dysfunction, and inflammation.