Bridge-like lipid transfer proteins (LTPs), characterized by repeating β-groove domains and extended hydrophobic grooves, form physical bridges at membrane contact sites (MCSs) to mediate efficient lipid exchange. Atg2 is one such bridge-like LTP essential for autophagosome formation, during which the isolation membrane (IM) emerges and expands through continuous lipid supply from the endoplasmic reticulum (ER). However, direct evidence for Atg2-mediated lipid transfer in vivo has been lacking. Here, we characterize octadecyl rhodamine B (R18) as a lipophilic dye that demonstrates non-vesicular transport. We find that R18 is internalized via type IV P-type ATPase flippases at the plasma membrane, and subsequently transferred to the ER by oxysterol-binding protein-related proteins. Finally, it reaches each autophagy-related structure (ARS), including the IM, autophagosome, and autophagic bodies, following the induction of autophagy. Using R18, we confirm that IM staining depends on Atg2 and perform fluorescence recovery after photobleaching (FRAP) assays that demonstrate rapid, consistent lipid flux at ER–IM MCSs, supporting an active lipid transfer model. Time-course tracking shows that the IM expands dynamically to enclose cargo, while the ER serves as the main donor membrane. Furthermore, upon terminating autophagy, we observe fluorescence recovery in the ER coupled with a decrease in IM signal, revealing back-transfer of lipids from the matured IM to the ER. Time-course tracking shows the back-forward lipid transfer from IM to ER and a dissociation of matured IM after stopping autophagy. Taken together, our findings suggest reversible unidirectional lipid dynamics via bridge-like LTPs at MCSs in vivo and highlight the critical role of bridge-like LTPs in MCS-mediated phospholipid homeostasis. We believe that it offers a great chance to further explore the conserved lipid transfer model at different MCSs in the future.