Chloroquine (CQ) is evaluated as autophagy-blocker for cancer treatment, but it is unknown if it solely acts by inhibiting cancer cell autophagy. We report that CQ reduced tumor growth but improved the tumor microenvironment. By normalizing tumor vessel structure and function and increasing perfusion, CQ reduced hypoxia, cancer cell invasion and metastasis, while improving chemotherapy delivery and response. Inhibiting autophagy in cancer cells or endothelial cells (ECs) failed to induce such effects. CQ’s vessel normalization activity relied mainly on alterations of endosomal Notch1 trafficking / signaling in ECs and was abrogated by Notch1 deletion in ECs in vivo. Thus, autophagy-independent vessel normalization by CQ restrains tumor invasion and metastasis while improving chemotherapy, supporting the use of CQ for anti-cancer treatment.