The purpose of this study was to determine both the short-term effects on cardiac development and embryo growth and the long-term effects on cardiac function and body composition of in utero caffeine exposure. Pregnant mice (C57BL/6) were exposed to hypoxia (10% O₂) or room air from embryonic days (E) 8.5–10.5, and treated with caffeine (20 mg/kg, i.p.) or vehicle (normal saline, 0.9% NaCl). This caffeine dose results in a circulating level that is equivalent to 2 cups of coffee in humans. Hypoxic exposure acutely reduced embryonic growth by 30%. Exposure to a single dose of caffeine inhibited cardiac ventricular development by 53% in hypoxia and 37% in room air. Caffeine exposure resulted in inhibition of hypoxia-induced HIF1 protein expression in embryos by 40%. When offspring from dams treated with a single dose of caffeine were studied in adulthood, we observed that caffeine treatment alone resulted in a decrease in cardiac function of 38%, as assessed by echocardiography. We also observed a 20% increase in body fat with male mice exposed to caffeine. Caffeine was dissolved in normal saline, so it was used as a control. Room air controls were used to compare to the hypoxic mice. Exposure to a single dose of caffeine during embryogenesis results in both short-term effects on cardiac development and long-term effects on cardiac function.—Wendler, C. C., Busovsky-McNeal, M., Ghatpande, S., Kalinowski, A., Russell, K. S., Rivkees, S. A. Embryonic caffeine exposure induces adverse effects in adulthood.