Global antimicrobial resistance has led to a ban on the use of antibiotics as growth promoters (AGPs) in poultry farming, encouraging the use of natural phytogenic feed additives that provide similar effects to AGPs without causing resistance. The aim of this study was to determine the effects of the addition of encapsulated fennel seed (Foeniculum vulgareMill.) essential oil (FEO) into the diets on the performance, intestinal microflora, morphology, and transcriptomic profiling of broiler chickens. In the study, 400 one-d-old male chicks of the Ross-308 genotype were randomly distributed into five groups, each with 16 replicates of five birds. The experiment included a control group fed on basal diets without the addition of FEO and treatment groups supplemented with 50 (FEO50), 100 (FEO100), 200 (FEO200), or 400 (FEO400) mg of encapsulated FEO/kg. Body weight and the European Production Efficiency Factor values were higher in the FEO100, FEO200, and FEO400 groups (P< 0.05). The feed conversion ratio significantly improved at all FEO levels (P< 0.05). FEO supplementation improved duodenum, jejunum, and ileum morphologies. It enhanced mucosal layer thickness in the duodenum and jejunum, and muscular layer thickness in the jejunum and ileum (P< 0.05). It also increased the number of Lactobacillusspp. in the jejunum and ileum (P< 0.05). According to the transcriptome profile obtained from the microarray analysis of samples taken from small intestine tissues, the mRNA expression levels of 261 genes in the FEO50 group (206 upregulated and 55 downregulated), 302 genes in the FEO100 group (218 upregulated and 84 downregulated), 292 genes in the FEO200 group (231 upregulated and 61 downregulated), and 348 genes in the FEO400 group (268 upregulated and 80 downregulated) changed compared to the control group. Most upregulated genes were associated with catalytic activity, binding, transcription regulators and transcription factors, anatomical structure and cellular development, and protein binding activity modulators. The downregulated genes mostly belonged to the transporter, carrier, and protein-modifying enzyme classes. Besides, the anti-inflammatory IL-10 gene (4.41-fold) increased significantly in the FEO100 group compared to the control group (P< 0.05). In conclusion, FEO improved the performance of broiler chickens by regulating biological processes such as performance and intestinal health, with the 100 mg FEO/kg supplementation being the most prominent.The optimal level of 100 mg encapsulated fennel seed essential oil per kilogram of diet significantly boosted growth, gut health, and transcriptomic responses in broiler chickens.The long-term use of low-level antibiotics in the poultry industry to promote growth, rather than for treatment, has led to the development of antimicrobial resistance (AMR), a significant risk to human and animal health. This concern has led to a ban on antibiotics as growth promoters (AGPs) in broiler diets, resulting in an increase in metabolic diseases in broiler chickens. Phytogenic feed additives have been tested as alternatives to compensate for the loss of AGPs. One such additive, fennel seed essential oil (FEO), known for its strong antimicrobial properties, was examined in this study for its impact on broiler growth performance and the underlying genetic and molecular mechanisms. The results showed that FEO enhanced feed conversion efficiency, thereby improving broiler performance. It positively affected the intestinal wall structure and function and helped establish a balanced microbiota by suppressing harmful and supporting beneficial microorganisms. Transcriptome datasets revealed that FEO modulated gene expression related to economically important traits such as performance and intestinal health. In conclusion, considering the urgent need for antibiotic-free production systems owing to escalating AMR and the growing interest in genotype-specific feeding in the postgenomic era, FEO may be a promising, natural, safe, and effective alternative to AGPs.