Rab7 and Rab9 are small GTPases associated with late endosomes. Rab7 localizes to lysosomes, where it facilitates vesicle fusion, while Rab9 primarily regulates endosomal trafficking. Recent studies have identified Rab9 as a critical regulator of Atg5/Atg7-independent alternative autophagy. However, the molecular relationship between Rab7 and Rab9 in the alternative autophagy remains largely unknown. Here, we investigated the role of Rab7 in Rab9-dependent alternative autophagy. We first found that Rab7 influences the subcellular localization of Rab9. Overexpression of a dominant-negative form of Rab7 led to increased accumulation of Rab9 at the Golgi apparatus and decreased localization to lysosomes, as shown by confocal microscopy. Using an AI-based prediction model, we identified the Rab7 effector PLEKHM1 as a potential adaptor for Rab9. Co-immunoprecipitation assays confirmed an interaction between Rab9 and PLEKHM1. Under amino acid starvation, colocalization of Rab9 and PLEKHM1 on lysosomes was markedly enhanced, whereas this colocalization was significantly reduced in cells expressing dominant-negative Rab7. To further assess Rab9-dependent autophagic flux, we established a Halo Tag-based assay system. In this system, Rab7 inhibition resulted in reduced levels of cleaved Halo Tag on Western blot, indicating that Rab9-mediated autophagic flux is compromised in the absence of Rab7 activity. Collectively, these findings demonstrate that Rab7 plays a crucial role in regulating Rab9-dependent alternative autophagy. Specifically, Rab7 modulates Rab9-dependent autophagy by influencing the interaction between Rab9 and PLEKHM1, uncovering a novel regulatory mechanism for vesicle fusion in alternative autophagy.