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BACKGROUND Stevioside (STE) is a widely used sweetener. Despite the fact that chickens are insensitive to sweetness, dietary STE supplementation could increase the feed intake of broiler chickens. STE might regulate the feeding behavior through other functional mechanisms rather than its high-potency sweetness. The present study was aimed to elucidate the potential sweetness-independent mechanism of STE-induced orexigenic effect using the broiler chicken as the animal model from the point of hypothalamic transcriptome profile and gut microbiome. RESULTS The analysis of RNA-Seq identified 398 differently expressed genes (160 up-regulated and 238 down-regulated) in the hypothalamus of the STE supplemented group compared with the control group. Cluster analysis revealed several appetite-related genes were differentially expressed, including NPY, NPY5R, TSHB, NMU, TPH2 and DDC. In addition, the analysis of 16S rRNA sequencing data indicated that dietary STE supplementation increased the relative abundance of Lactobacillales, Bacilli, Lactobacillus and Lactobacillaceae. Meanwhile, the proportion of Ruminococcaceae, Lachnospiraceae, Clostridia and Clostridiales was decreased after dietary supplementation with STE. CONCLUSION Dietary STE supplementation promoted the feed intake through the regulation of hypothalamic neuroactive ligand-receptor interaction pathway and the alteration of intestinal microbiota composition. This study provides valuable information about the sweetness-independent mechanism of STE-induced orexigenic effect using the broiler chicken (insensitive to sweetness) as the animal model. This article is protected by copyright. All rights reserved.