ABSTRACT Cardiovascular disease (CVD) is the leading cause of mortality, accounting for over a third of all deaths globally. CVD is caused by atherosclerosis, a complex chronic inflammatory disorder that results from chronic damage to the arterial wall by inflammation and lipid accumulation. Atherosclerosis begins in early life and is essentially universal by early adulthood. Thus, there is broad consensus that CVD prevention should begin early when adverse trajectories may be most modifiable – potentially as early as during infancy. Epidemiological data show that breastfed infants are at lower risk of developing CVD later in life, however, the protective components in human breastmilk and underlying mechanisms that contribute to the beneficial effects of breastfeeding remain unknown. After lactose and lipids, human milk oligosaccharides (HMOs), a group of complex carbohydrates, are the third most abundant component of human breastmilk. HMOs – either directly, or indirectly through shaping the infant gut microbiome – can impact infant inflammation and lipid metabolism, the two key contributors to atherosclerosis and CVD. In fact, our preliminary data shows that mice that received the HMO 3’-sialyllactose (3’SL) with the dam’s milk during the breastfeeding period had significantly lower plasma triglyceride and cholesterol levels and developed less atherosclerosis later in life. Here, we aim to explore whether these observed beneficial effects translate from mice to humans. Based on the finding that HMO composition varies between women and has been associated with several infant health and disease outcomes, we propose to test the hypothesis that HMO amount and composition associate with early preclinical markers of CVD and metabolic risk, and markers of inflammation from birth to early childhood. We will leverage the extraordinary resources of the Barwon Infant Study (BIS), one of the most comprehensive pre-birth cohort studies of cardiometabolic and inflammatory phenotypes in the world, apply state-of-the-art technology to measure HMO composition in biobanked breastmilk and infant plasma samples, and test whether HMO concentrations associate with infant lipidomic, metabolic, and cardiovascular phenotypes (AIM 1) as well as infant markers of inflammation (AIM 2). Knowledge gained from the successful completion of the proposed project will add to the existing preclinical data that established causal relationships. Together, the results will greatly enhance our understanding why breastfed infants are at lower risk of later CVD and form the basis for early life interventions. This is particularly timely as HMOs like 3’SL are now synthesized in bioengineered microbes and stand ready as new, inexpensive, scalable and safe options to help prevent CVD early in life when adverse trajectories may be most modifiable and help address the substantial and increasing health and economic costs of CVD.