Background/Objectives: The forkhead factor Foxa3 is involved in the early transcriptional events controlling adipocyte differentiation and plays a critical function in fat depot expansion in response to high-fat diet regimens and during aging in mice. No studies to date have assessed the potential associations of genetic variants in FOXA3 with human metabolic outcomes. Subjects/Methods: In this study, we sequenced FOXA3 in 392 children, adolescents and young adults selected from several cohorts of subjects recruited at the National Institute of Child Health and Human Development of the National Institutes of Health based on the availability of dual-energy X-ray absorptiometry data, magnetic resonance imaging scans and DNA samples. We assessed the association between variants present in these subjects and metabolic traits and performed in vitro functional analysis of two novel FOXA3 missense mutations identified. Results: Our analysis identified 14 novel variants and showed that the common single-nucleotide polymorphism (SNP) rs28666870 is significantly associated with greater body mass index, lean body mass and appendicular lean mass (P values 0.009, 0.010 and 0.013 respectively). In vitro functional studies showed increased adipogenic function for the FOXA3 missense mutations c.185C > T (p.Ser62Leu) and c.731C > T (p.Ala244Val) compared with FOXA3-WT. Conclusions: Our study identified novel FOXA3 variants and mutations, assessed the adipogenic capacity of two novel missense alterations in vitro and demonstrated for the first time the associations between FOXA3 SNP rs28666870 with metabolic phenotypes in humans.