Autophagy is essential for cell survival and cellular homeostasis under various stress conditions. Therefore, autophagy dysfunction is associated with the pathogenesis of various human diseases. We explored the regulatory role of RhoBTB3 in autophagy and its interaction with activating molecules in AMBRA1. RhoBTB3 deficiency was found to induce autophagy, while its overexpression inhibited autophagy induction. Through immunoprecipitation and mass spectrometry, AMBRA1 was identified as a substrate of RhoBTB3. The study revealed that RhoBTB3 regulates AMBRA1 stability by influencing its protein levels without affecting its mRNA levels. RhoBTB3 induced the ubiquitination of AMBRA1, leading to proteasome-mediated degradation, with the ubiquitination occurring at K45 on AMBRA1 through a K27-linked ubiquitin chain. The knockdown of AMBRA1 blocked RhoBTB3 knockdown-induced autophagy, indicating the dependency of autophagy on AMBRA1. Thus, RhoBTB3 negatively regulates autophagy by mediating AMBRA1 ubiquitination and degradation, suggesting RhoBTB3 as a potential therapeutic target for autophagy-related diseases.