Stunting, a common form of chronic undernutrition, is associated with impaired nutrient absorption and glucose transport. Hibiscus sabdariffa Linn. (roselle) contains bioactive flavonoids and anthocyanins that have the potential to regulate glucose transporter 1 (GLUT1). This study aimed to examine the effect of H. sabdariffa phytochemicals on GLUT1 regulation in the intestine, using the HT29 cell line as a model. An integrated approach combining in silico docking and in vitro validation was employed. Seven key phytochemicals were docked to GLUT1 using AutoDock Vina. Cytotoxicity was evaluated by MTT assay, and GLUT1 mRNA expression was analyzed by qPCR after treatment with sub-cytotoxic concentrations (≤ IC₅₀ = 17.2 µg/mL) for 24 and 48 hours. The results of the study revealed that among the isolated chemicals, Delphinidin-3-sambubioside and cyanidin-3-sambubioside showed the highest affinities (–10.3 and –9.8 kcal/mol, respectively). Treatment with H. sabdariffa resulted in a significant downregulation of GLUT1 expression to 0.68 ± 0.09-fold (p < 0.05) after 24 hours and 0.42 ± 0.07-fold (p < 0.01) after 48 hours. These findings indicate that H. sabdariffa phytochemicals have the potential to modulate intestinal glucose transport by suppressing GLUT1 expression. This research provides initial insights into the regulation of nutrient uptake through phytochemicals, which may be relevant to the mechanisms underlying stunting. © 2025, Editorial board of Journal of Experimental Biology and Agricultural Sciences. All rights reserved.