Background Acidifier are substances with antibacterial, antifungal, antimicrobial, performance and health benefits that are frequently employed in feed acidification, especially in poultry diet. Meanwhile, the most important factor for acidifier efficiency is the proportion of different acids in the final product. Objectives This study aimed to investigate the effect of dietary supplementation of a commercial acidifier on egg production and histology of the small intestine in laying Japanese quail. Methods One‐hundred and sixty female quails at 15 weeks of age were divided into four groups and fed basal diet supplemented with different levels of acidifier (0, 1, 2 and 3 gr acidifier/kg of basal diet) for 8 weeks. Egg production, egg quality attributes and body weight (BW) were measured every 2 weeks. Histology of the small intestine and bacterial population of cecum as well as pH of crop, duodenum, jejunum, ileum and cecum contents were also investigated at the end of the experiment. Results Feed conversion ratio (FCR), yolk height, shell thickness, pH of the duodenum, jejunum, ileum, cecum; duodenum, villus width (VW), villus height (VH), crypt depth (CD); jejunum VH, VW and ileum VH to CD ratio (VCR) were linearly improved by the increasing levels of acidifier supplementation ( P < 0.05). Duodenum VH increased in a linear and quadratic manner in response to increasing levels of acidifier. Egg weight, yolk diameter, jejunum CD, ileum CD, ileum VW, duodenum CD and jejunum VCR quadratically improved by grading levels of acidifier ( P < 0.01). BW, albumen height, Haugh unit, ileum VH and ileum VCR were cubically enhanced ( P < 0.05). Acidifier supplementation enhanced egg production, FCR, jejunum, ileum and cecum pH and VH, CD and VW of duodenum and jejunum, compared to the control group ( P < 0.05); however, dietary acidifier did not affect egg mass, gizzard pH, ileum VH and bacterial count of the cecum ( P > 0.05). Conclusions In conclusion, as calculated, the supplementation of 1 and 2.6 g acidifier per kg of diet was associated with beneficial effects on egg production and quality, gastrointestinal tract pH and histology of the small intestine in laying quails.