Early life stress (ELS) is linked to risks for later-life disorders with a sex bias (e.g., depression, obesity, and Alzheimer’s disease). It remains unclear whether such bias has early origins and if sex-dependent adaptations to ELS play a role. We focused on the hypothalamus, a crucial neuroendocrine stress axis (Hypothalamus-Pituitary-Adrenal axis) modulator, and studied gene expression profiles under control conditions and how ELS affects these long-term, and how sex and ELS impact the acute stress response.Mice underwent ELS by limited bedding and nesting from postnatal day (P) 2-9, then acute stress by 30-minute restraint (RS) at P70. Bulk hypothalamic RNA sequence analysis with DESeq was done to identify differentially expressed genes (DEGs) (log2FC>0.1, FDR<0.05). Over-Representation Analysis (ORA) and Expression Weighted Cell-type Enrichment (EWCE) were done to predict associated processes and cell types.DEG analyses revealed that ELS affected gene expression at baseline in females only. Investigating the RS response, we found that expression of predominating immediate early genes (IEGs) was not influenced by ELS or sex (z-score, p>0.05). Probing further, IEG exclusion and use of more exploratory thresholds (log2FC>0.5, p<0.05) revealed that RS response is ELS- and sex-dependent. Additionally, neuronal processes were more overrepresented in male responses to ELS and ELS+RS versus females, and sex-related patterns were observed in enriched cell type profiles in ELS, RS, and ELS+RS exposure.For protein expression validation by immunohistochemistry, parvalbumin (Pvalb) and oxytocin (Oxt) were identified from the top 50 (by log2FC) DEGs (p<0.05, CPM>10) in both sexes, regardless of stress exposure.