Objectives/Goals: Opioid use disorder (OUD) in pregnancy and its implications on the maternal-fetal interface has been relatively understudied. Here, we aimed to uncover the impact of maternal OUD on placental structure, function, and inflammatory responses and further stratified our findings by maternal hepatitis C (HCV) infection. Methods/Study Population: To address this knowledge gap, we collected placental tissue from healthy pregnancies (control) and those with opioid use disorder with and without maternal HCV infection. First, placental development was assessed by gross and histological examination of the placenta. Immune cells were then isolated from decidua (maternal) and chorionic villous (fetal) placental tissues, and the frequency and phenotype of immune subsets were determined by flow cytometry. Markers of inflammation, placental perfusion, growth factors, tissue remodeling, and vascularization were measured in placental tissue homogenate by multiplex Luminex assay. Finally, gene expression alterations in placental architecture were assessed by Visium spatial transcriptomics, integrating transcriptomic data with spatial information. Results/Anticipated Results: Our results indicate that maternal OUD impairs placental perfusion/development and is accompanied by increased markers of inflammation in the decidua (IL-1Ra, IL-2, IL-18, IP-10, MIP-1β, and TNFα) and villous (IL-6 and IL-8). Furthermore, markers of angiogenesis and placental development are altered in the decidua, including increased EGF and IL-6Ra, but decreased FLT-1, FLT-4, and bFGF. The abundance of placental immune cells is varied with OUD/HCV, including decreased frequencies of decidual macrophages and NK cells, critical for blood supply to the fetus, and increased abundance of infiltrating maternal macrophages in fetal chorionic villous. Finally, spatial transcriptomics revealed aberrant infiltration of activated immune cells and modified processes associated with inflammation and angiogenesis. Discussion/Significance of Impact: Altogether, these findings suggest a profound impact of maternal OUD with and without maternal HCV infection on the structure, function, and immune landscape of the maternal–fetal interface that can alter fetal development and maturation.