Abstract Background: In postpartum dairy cows, the energy needs to satisfy high milk production induces a more or less pronounced Negative Energy Balance (NEB) status. NEB associated with fat mobilization impairs reproductive function. This study investigated the specific impact of NEB on gene expression in the three main types of endometrial cells at time planned for insemination and implantation. Endometrial cell types (stromal, glandular and luminal epithelial cells) were isolated by laser micro-dissection allowing the study of constitutive gene expression and their specific response to NEB. Methods: Nine Swedish Red cows receiving a control diet or a mild restricted diet to induce differences of energy balance were categorized into mild (MNEB, n = 5) and severe negative energy balance (SNEB, n = 4). The three endometrial cell types: luminal (LE), glandular (GE) epithelium and stroma (ST) were collected by laser microdissection from endometrial biopsies performed at 80 days postpartum. Results: Transcriptome profiles obtained by RNA sequencing revealed differences in constitutive gene expression between the three cells types and also differences in specific responses related to the severity of NEB. Number of differentially expressed genes between SNEB and MNEB cows was higher in ST than in LE and GE, respectively. SNEB was associated with differential expression of genes related to metabolic processes and embryo-maternal interactions in ST. Under-expression of genes related to cell structure was found in GE whereas genes related to pro-inflammatory pathways were over-expressed. Genes associated to adaptive immunity were under-expressed in LE. Conclusion: The three different main cells types of the endometrium, have very different patterns of gene expression. The severity of NEB after calving is associated with changes in gene expression at time of breeding. Specific alterations in GEs are associated with activation of pro-inflammatory mechanisms. Concomitantly, changes in the expression of genes related to cell to cell interactions and maternal recognition of pregnancy takes place in ST. The combination of these effects possibly altering the uterine environment and embryo maternal interactions may negatively influence the establishment of pregnancy.