Updated project metadata. Extracellular vesicles (EV) are signaling entities that are released by most, if not all, eukaryotic cells. EV are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating invasion. Macroautophagy is a catabolic process initially known for the recycling of cytosolic cargos through lysosomal degradation, while its non-degradative functions have only begun to emerge. To better understand the relationships between autophagy machinery and small EV (sEV) biogenesis, we utilized chloroquine (CQ) to inhibit lysosomal degradation and autophagy turnover in triple-negative breast cancer (TNBC) cell lines and characterized its effects on sEV content and function. We performed quantitative mass spectrometry analysis of the sEV proteome, and discovered a CQ-induced enrichment of mammalian ATG8 paralogs and their adaptor proteins that coincided with increased levels of K48-specific polyubiquitination in sEV, as well as with the co-localization of ATG8s and endolysosomal markers in the cytoplasm. Using ATG4B knockout cells, we established the lipidation-dependent luminal localization of sEV-associated ATG8s. We demonstrated CQ-mediated enrichment of MAP1LC3B and GABARAP in CD63-high but not CD9-high sEV subpopulations. sEV isolated from CQ treated versus untreated cells had differential effects on recipient cell growth and HMEC-1 tube formation, suggesting sEV are an avenue of CQ mediated non-cell-autonomous biological effects. Our study demonstrates the flexibility of sEV composition in response to perturbation of intracellular trafficking pathways, and has implications for CQ efficacy in therapeutic settings.