Intravesical　chemotherapy　is　generally　used　in　the　clinic　for　treating　bladder　cancer　(BCa),　but　its　efficacy　is　limited　due　to　the　permeation　barrier　and　side　effects　caused　by　the　off-targeting　of　normal　urothelial　cells.　In　this　study,　BCa　cell-derived　membrane　nanovesicles　were　used　as　drug　carriers,　and　their　homologous　tumor-targeting　capacity　was　utilized.　A　BCa-targeting　hendeca-arginine　peptide　was　functionalized　onto　the　nanovesicles　to　impart　a　mucus-penetrating　ability　and　thus　overcome　the　permeation　barrier.　The　tumor-targeting　and　mucus-penetrating　nanovesicles　were　stable　in　urine,　were　highly　permeable　to　the　glycosaminoglycan　layer,　and　specifically　targeted　BCa.　The　vesicles　were　internalized　through　caveolin-mediated　endocytosis,　were　transported　to　nonlysosome-localized　intracellular　regions,　and　efficiently　infiltrated　bladder　tumor　spheroids.　In　in　vivo　intravesical　chemotherapy,　the　nanovesicles　achieved　chemo-resection　in　murine　orthotopic　BCa　models.　This　BCa-targeting　and　mucus-penetrating　drug　delivery　system　may　be　promising　for　the　intravesical　chemotherapy　of　BCa.　©　2022　The　Authors