In 1999, candidate gene screening within Anishininew (Oji-Cree) communities uncovered a novel genetic variant in the HNF-1α gene (HNF-1αG319S). Hailed as the strongest genetic predictor of type 2 diabetes (T2D) ever identified, 40% of diagnosed Manitoban youth were also found to be carriers of this variant “S-allele”. The newly discovered G319S variant was believed to drive pancreatic beta-cell dysfunction; however, youth-onset T2D is a relatively recent phenomenon. Therefore, we hypothesized that the G319S variant impairs insulin secretion when exposed to dietary carbohydrate stress but is protective in the context of traditional, off-the-land foods that are rich in fat and protein. To examine this relationship, CRISPR-Cas9 was used to knock-in the G>A.955 substitution into clonal MIN6 beta-like cells (“G319S-MIN6”) and into a C57BL/6N mouse background generating two novel experimental models. Mice were weaned onto (1) standard grain-based chow, (2) a high-fat, low-carbohydrate (HFLC) diet modelled after traditional, off-the-land foods, or (3) a high-fat, high-carbohydrate (HFHC) diet reflecting present-day Western dietary patterns. In vitro, we assessed gene expression, insulin secretion dynamics, and oxidative respiration under different macronutrient exposures. In vivo, we completed a comprehensive characterization of both male and female mice between 3- and 6-months-of-age terminating with islet isolation to assess insulin secretion and gene expression by single-cell RNA sequencing. In support of our hypothesis, the short-term consumption of a HFLC diet appears to normalize insulin secretion and glucose tolerance whereas a HFHC diet accelerates cardiometabolic dysfunction and impairs insulin secretion in G319S-expressing mice. In G319S-MIN6 cells or islets, fatty acid oxidation was increased, and gene expression changes supported a metabolic switch away from glucose respiration. Importantly, these findings demonstrate that genetics alone cannot explain T2D onset in G319S-carriers, rather gene-diet interactions, particularly a Westernized diet containing simple carbohydrates and saturated fats, leads to distinct metabolic outcomes that impact short- and long-term health. These studies may inform nutritional interventions for youth with T2D while ultimately supporting community-led efforts to access off-the-land traditional foods.