BACKGROUND: Numerous studies have investigated the positive effect of chitosan and nano-chitosan loaded clinoptilolite on growth performance, digestive enzyme activity, and intestinal histomorphology in different fish species. However, there are no data evaluating the potential effect of the composites on the fish stomach.OBJECTIVES: In the current study, the effects of chitosan and nano-chitosan loaded clinoptilolite on histological features and pepsin activity in the rainbow trout stomach were considered.METHODS: Chitosan and nano-chitosan loaded clinoptilolite were synthesized, and then two hundred and forty fish (~27.75 g) fish were distributed in eight groups each in three replicates. Ten days after adaptation, the fish were fed with eight diets, including control diet (CTR), clinpotilolite (T1), chitosan composites (T2, T3, T4), and nano-chitosan composites (T5, T6, T7) for 70 days. Afterward, all fish in each tank were anesthetized in clove extract (50 μl/l), and tissue samples were obtained for pepsin activity (n= 5) and histological assay (n = 5).RESULTS: The groups administrated with nanochitosan composites showed the highest pepsin activity (P˂0.05). Additionally, histological examinations exhibited a higher epithelial height, increased mucosal density, and oxynticopeptic cells hypertrophy in fish fed composites compared to the CTR group (P˂0.05). Meanwhile, nanochitosan composite administration could cause higher reaction of secreted granules to periodic acid–Schiff (PAS) staining.CONCLUSIONS: The findings demonstrated the potential application of chitosan and nano-chitosan loaded clinoptilolite composites for improvements in the histomorphology and pepsin activity of the rainbow trout stomach, resulting in higher growth performance.