Background/Aims: While maternal stress has been linked to adverse child health outcomes, mechanistic links have not been fully elucidated. Maternal microRNAs encapsulated in Extracellular Vesicles (EVs) reach the infant through breastmilk and are a novel biochemical communication pathway for early-life programming. We leverage the PRogramming of Intergenerational Stress Mechanisms (PRISM) pregnancy cohort to investigate associations between maternal stress and breast milk EV-microRNAs. Methods: We assessed maternal lifetime stress using the Life Stressor Checklist-Revised (LSCR) survey and negative life events (NLEs) experienced in the past 6 months during pregnancy using the Crisis in Family Systems-Revised (CRISYS-R) survey. Extracellular vesicles were isolated from N=80 breastmilk samples collected at 6.1±5.9 weeks postnatally. Total RNA was extracted and microRNAs were profiled using the TaqMan OpenArray Human miRNA panel. Logistic regression assessed associations between continuous LSCR and NLE scores and EV-microRNA detection (outcome yes/no);associations with EV-microRNA expression levels were assessed using robust linear regression (N=74 with EV-microRNA and maternal stress data). Models were adjusted for infant sex, maternal race/ethnicity, education, and week of breast milk collection. Results: Among 345 EV-microRNAs detected in >10% of samples, detection of 127 (47%) was associated with LSCR score and detection of 97 (28%) was associated with NLE score (p < 0.05). Among 205 EV-microRNAs detected in >50% of samples, expression of 8 was associated with LSCR scores and expression of 17 was associated with NLE score at our a priori criteria of p < 0.05 and |Bregression| > 0.2. MicroRNAs associated with LSCR and NLE scores were involved in KEGG pathways related to fatty acid metabolism and steroid biosynthesis. Conclusions: Maternal lifetime cumulative stress and stress during pregnancy were associated with EV-microRNAs in breast milk although microRNA profiles differed. Further research is needed to identify biological pathways impacted by differentially expressed microRNAs and investigate relationships with child health outcomes.