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Objective This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood. Study Design Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid (n = 13) and cord blood samples (n = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health. Results Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples. Conclusion Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health. Key Points NNS consumption during pregnancy has increased in recent years. Maternal NNS intake during pregnancy is associated with preterm birth and higher infant weight gain in epidemiologic studies. In rodents, in utero NNS exposure induces metabolic abnormalities in mothers and their offspring, alters offspring gut microbiota composition, and promotes sweet taste preference in adulthood. It is presently unknown whether and to what degree maternal NNS ingestion in humans leads to direct in utero exposure. This study provides the first evidence of in utero NNS exposure in humans and highlights the urgent need to investigate clinical consequences of early life NNS exposure on metabolism, weight, taste preference, and general health. [ABSTRACT FROM AUTHOR]