Hypothalamus integrates environmental and peripheral signals to coordinate and manage growth, reproduction, feeding, sleeping and behavior functions. And its transcriptomic profile changes are based on body physiological status. A good way for bird requirement calculation is associating those transcription alterations in hypothalamus for transition from laying to incubating state and vice versa in poultry. Accordingly RNA-Seq data of hypothalamus gene expression were downloaded from NCBI (http://www.ncbi.nlm.- nih.gov/sra/DRA008396). The quality control of sequencing reads was carried out by FastQC software. Filtered reads were evaluated with FastQC. The transcriptional profiling of 4 hypothalamus Sampels including 2 samples from broodiness and 2 samples from non-broodiness was assessed using DEseq2 Package .Gene ontology and pathway analysis were performed using bioinformatics tools. String database was used for the retrieval of interacting genes and Cytoscape software was used to visualize the network. In hypothalamus of laying hens comparing to incubating hens 17 genes showed significant expression change. Eight down-regualtion was related to serotonin receptor 4 (HTR4), prolactin receptor (PRLR), prolactin (PRL), Aguti-related protein (AGRP), Ghrelin (GHRL), neuro-peptide receptor 1 and 2 (NPY1R and NPY2R), vaso-active intestinal peptide (VIP) and nine up-regulation was related to gene expression of melatonin receptor 1 (MTNR1), Arginine vasotocin (AVP), Prolactin Releasing Hormone (PRLH), growth hormone releasing hormone (GHRH), Thyroid releasing hormone (TRH), orexin (HCRT), Pro-Melanin Concentrating Hormone (PMCH), tryptophan hydroxylase 1 and 2 gene expression (TPH1 and TPH2). In conclusion, although pro-lactogenic pathway declined significantly but serotonergic and melatonergic pathways related genes proliferated in hypothalamus of laying hen. AGRP gene and specially ghrelinergic pathway, VIP gene decreased. Accordingly it might to be told more supplementation of tryptophan amino acid and its melatonergic metabolites during laying period is necessary.