Objective: This study investigated the effects of Apidaecin Api-PR19 as feed additive on growth performance, intestinal health, and small intestinal microbiota of broilers.Methods: A total of 360 1-d-old Arbor Acres broilers were randomly assigned to 3 groups with 6 replicates including control group with basal diet (CON), antibiotic growth promotor group with basal plus 10 mg/kg colistin sulfate and 50 mg/kg roxarsone (AGP), and antibacterial peptide group with basal diet plus 330 mg/kg Apidaecin Api-PR19 (ABP). The trial lasted 35 d.Results: Results showed that dietary Api-PR19 addition increased (p<0.05) the average daily feed intake, average daily gain and decreased (p<0.05) feed conversion ratio (FCR) during 1 to 21 d compared with the CON group. The digestibility of dry matter and crude protein were higher in AGP and ABP groups (p<0.05) where greater trypsin activity was detected in duodenum (p<0.05). The ratio of villus height to crypt depth (V/C) in duodenum and jejunum was increased at 35 d when broilers were given diets with ABP or AGP (p<0.05). Besides, ABP treatments up-regulated (p<0.05) the mRNA expression of <i>EAAT3, GLUT2, ZO-1</i>, and <i>Claudin-1</i> in duodenum of broilers at 35 d of age. The results of immunohistochemistry showed that ABP treatment significantly increased (p<0.05) duodenal secretory immunoglobulin A (sIgA) content. In addition, 16S rRNA gene sequencing revealed that there were differences in the intestinal microbiota diversity and composition among three groups. Notably, the linear discriminant analysis effect size showed that <i>p_Firmicutes, g_Enterococcus, g_Carnobacterium, g_Kitasatospora</i>, and <i>g_Acidaminococcus</i> were dominant in ABP group. Redundancy analysis showed that these changes in gut microbiota in ABP group had correlation with growth performance, intestinal morphology, and content of sIgA.Conclusion: In general, these results indicated that dietary 330 mg/kg Apidaecin Api-PR19 supplementation promoted growth performance of broilers by improving intestinal development, nutrients absorption, immune function and modulating intestinal microbiota.