Palmitic acid (PA) is the main saturated fatty acid naturally occurring in animal fats and vegetable oils. In recent decades, palm oil, an alternative lipid source containing high amounts of PA, has been widely used to replace fish oil in aquafeed.,We investigated the hepatotoxicity of PA in zebrafish and the underlying mechanism.,One-month-old zebrafish fed a high-fat diet (HFD) containing 16% soybean oil and 3 PA-incorporated HFDs [4%, 8%, and 12% PA (12PA)] for 2 wk (experiment 1) and 4 wk (experiment 2) were used to evaluate PA-induced liver damage and endoplasmic reticulum (ER) stress. Germ-free (GF) zebrafish fed low-fat, high-fat, or 12PA diets for 5 d were used to study the direct effects of PA on liver damage (experiment 3). GF zebrafish colonized with HFD or 12PA microbiota for 48 h were used to elucidate the indirect effects of PA-altered microbiota on liver damage (experiment 4). Last, GF zebrafish colonized with HFD or 12PA microbiota were used to evaluate the effects of different microbiotas on PA absorption (experiment 5).,In experiment 1, the proportion of PA in the liver linearly increased as its percentage in dietary lipid increased (r2?=?0.83, P?