With the extensive use of plastic products, significant amounts of microplastics, nanoplastic particles (NPs), and plasticizers such as Di(2-ethylhexyl) phthalate (DEHP) are continuously released into the environment. However, the toxic effects of NPs alone or in combination with DEHP on mammary glands remain unreported. This study investigates the impacts of NPs and DEHP on the structure and function of mouse mammary epithelial cells and elucidates the underlying molecular mechanisms. We found that co-exposure to NPs and DEHP induced severe pyroptosis, inflammation and oxidative stress in HC11 cells. Co-exposure also caused mitochondrial damage, as evidenced by changes in mitochondrial membrane potential, increase in mitochondrial ROS and inhibition of ATP production. Moreover, NPs and DEHP co-exposure increased the transcriptional levels of endoplasmic reticulum (ER) stress-related genes, activated the inflammation-related NLRP3 signaling pathway, and damaged the cell membrane integrity. Notably, Co-exposure enhanced the ER-mitochondria crosstalk in HC11 cells, as evidenced by the upregulated transcriptional levels of ER Ca2+ channel proteins (Ip3r1, Grp75 and Vdac1), increased mitochondrial Ca2+ levels, and expanded mitochondrial-ER contact areas. In summary, this study revealed that NPs and DEHP co-exposure had the potential to induce pyroptosis and inflammation by enhancing the ER-mitochondria crosstalk, ultimately resulting in injury to mammary glands. These findings would provide some new insights into the molecular mechanisms underlying the toxic effects of NPs and DEHP to mammary glands.