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The influence of dietary supplementation of Lactobacillus acidophilus, on growth performance, digestive enzyme activities, gut histomorphology and gut microflora were evaluated in juveniles striped catfish (Pangasianodon hypophthalmus). Five experimental diets were formulated by supplementing 0, 103, 105, 107 and 109 CFU/g L. acidophilus in fishmeal and casein‐based semi‐purified diet. Triplicate groups of striped catfish (21.69 ± 0.18 g) were stocked in 15 fiberglass tanks with stocking density of 25 individuals per tank and fed twice daily at 2.5% of the fish body weight for 12 weeks. Weight gain, specific growth rate, feed conversion ratio and the protein efficiency ratio were significantly higher in fish fed with 105 and 107CFU/g L. acidophilussupplemented diets compared with the other treatment groups. Compared with the control and fish fed low (103 CFU/g) L. acidophilus supplementation, those fed with 105 and 107 CFU/g had significantly higher (p < 0.05) apparent protein digestibility. Inclusion of L. acidophilus at 107 CFU/g diet significantly increased amylase, protease and lipase activities. Microscopic analysis showed that the villi length in both the anterior and posterior gut and microvilli length in the posterior gut increased significantly in fish fed L. acidophilus supplementation at 105 and 107 CFU/g of diet. The fish fed L. acidophilus supplemented diets significantly increased the total lactic acid bacteria counts in the gut of striped catfish compared with the control‐fed group. Based on gut histomorphology and growth performance, inclusion of L. acidophilus at 105 CFU/g appears to have the most positive effect on striped catfish farming. [ABSTRACT FROM AUTHOR]