<p>Autophagy is a cellular process essential for maintaining homeostasis and supporting development. Herein, we aimed to evaluate the effects of gestational protein restriction (GPR) on tooth germ autophagy. CAG-RFP-EGFP-LC3 female mice were mated and randomly assigned to normal protein or low-protein groups. Embryos were collected, and autophagy flux in the molar tooth germ was quantified. At the bud stage, GPR induced a decrease in autolysosomes in the condensed mesenchyme, alongside an increase in autophagosomes in the dental epithelium. At the cap stage, an increase in lysosome markers was observed in the dental papilla, while a decrease in autolysosomes was observed in enamel organ. By the bell stage, dental papilla exhibited a decrease in autolysosomes and an increase in lysosome markers. Similarly, inner enamel epithelium showed reduced autolysosomes and elevated lysosome markers. Bud stage was the most significantly affected; we further quantified apoptosis and mitosis, revealing impaired cell proliferation and enhanced apoptosis. Our data suggest that GPR block or delay acidification of autophagic vesicles, potentially impairing the recycling of cellular components critical for tooth germ proliferation and differentiation.</p> Graphical abstract <p></p>