Abstract The current study investigated the effects of stocking density (SD) on the performance, tibia mineralization, and the hypothalamic appetite genes expression in broilers. A total of 2,800 1-d-old male broilers (Cobb 500) were distributed in a completely randomized design to 1 of 5 SD treatments with 8 replicate cages for each treatment. The SD treatments were 12.5, 15.0, 17.5, 20.0, and 22.5 birds/m2, corresponding to 50, 60, 70, 80, and 90 birds per cage (4 m2/cage), respectively. The concentration of tibia phosphorus was determined by the ammonium metavanadate colorimetric method and the mRNA abundance in different tissues was measured by the real-time quantitative PCR method. The data were analyzed by the one-way and/or two-way analysis of variance and polynomial contrasts were used to determine the effect of increasing SD. Feed intake linearly decreased (P < 0.05) with increasing SD during d 1-42 production period. On d 42, body weight and tibia breaking strength were significantly lower in the groups of 17.5, 20.0 and 22.5 birds/m2 than in the groups of 12.5 and 15 birds/m2 (P < 0.01). Concentrations of ash and phosphorus in the tibia of broilers linearly decreased (P < 0.03) with increasing SD on d 42. The SD of 22.5 birds/m2 decreased the mRNA abundance of neuropeptide Y (NPY), NPY-receptor (NPYR) 1, and NPYR2 (P < 0.05), while it increased melanocortin receptor 4 mRNA abundance (P = 0.012) in the hypothalamus of broilers as compared with the SD of 12.5 birds/m2 on d 21 and 42. The mRNA abundance of hypothalamic cocaine and amphetamine-regulated transcript (CART), corticotrophin-releasing factor (CRF), and CRF-receptor 1 (CRFR1) were higher (P < 0.05) in the group of 22.5 birds/m2 than in the group of 12.5 birds/m2 on d 21. We concluded that increasing stocking density beyond 15 birds/m2 (corresponding to the 45 kg/m2 at 42 days of age) suppressed final BW and bone mineralization of broilers raised in multitier cage system. Hypothalamic NPY and CRF signaling might be involved in the anorexigenic effect of HSD.