Due to the substantial secretory burden, bovine mammary epithelial cells (BMECs) are highly susceptible to endoplasmic reticulum (ER) stress caused by the accumulation of misfolded proteins when protein-folding capacity is overwhelmed. However, how ATF3 regulates ER stress-induced impairment of milk synthesis and apoptosis in BMECs, particularly through its direct transcriptional targets, remains poorly understood. In this study, we investigated the protective role of activating transcription factor 3 (ATF3) against ER stress-induced impairment of milk synthesis in BMECs. Using a tunicamycin-induced ER stress model, we overexpressed ATF3 in BMECs and performed integrated RNA-seq and ChIP-seq analyses to elucidate the underlying molecular mechanisms. Our results indicated that ER stress disrupted milk protein and fat synthesis in BMECs by suppressing the expression of CSN2, FASN, FABP3 and promoting apoptosis via upregulation of BAX and CASP3. ATF3 overexpression effectively attenuated these effects, reducing apoptosis and restoring the expression of milk fat-related genes. Transcriptomics demonstrated that ATF3 activated MAPK and PI3K-Akt signaling and lipid metabolism pathways, significantly upregulating key genes involved in fatty acid uptake, transport, and metabolism (CD36, SLC27A1, ACSL1, PLIN1). Integrated RNA-seq and ChIP-seq analyses identified 81 overlapping genes, with RASGRP2, PRKACB, MAP3K5, and DUSP10 confirmed as direct transcriptional targets of ATF3, mediating its regulation of the MAPK pathway. Collectively, these findings elucidate the protective role of ATF3 against ER stress-induced lactation disruption and offer potential molecular targets for enhancing lactation resilience in dairy cattle under stress.