Novel poly(ethylene oxide)-graft-doxorubicin (PEO-g-DOX) prodrugs with DOX covalently conjugated to PEO via a pH-sensitive hydrazone bond were developed. PEO-g-DOX conjugates could be readily prepared in the following steps: (i) anionic ring-opening copolymerization of ethylene oxide (EO) and allyl glycidyl ether (AGE) afforded functional PEO with controlled molecular weights, low poly-dispersities, and multiple pendant double bonds (PEO-g-allyl); (ii) conjugation of PEO-g-allyl with methyl mercaptoacetate, followed by treating with hydrazine hydrate, quantitatively transformed allyl into hydrazide groups (PEO-g-hydrazide); and (iii) DOX was covalently immobilized to PEO-g-hydrazide via acid-labile hydrazone bonds (PEO-g-DOX). Here on the basis of PEO-g-allyl(4.4) (M-n GPC = 22 400, PDI = 1.19) and PEO-g-allyl(7.1) (M-n GPC = 15300, PDI = 1.16, the subscription refers to number of allyl groups per chain) two freely water-soluble PEO-g-DOX prodrugs with 2,9 and 3.6 DOX per molecule (denoted as PEO-g-DOX2.9 and PEO-g-DOX3.6, corresponding to drug loading content of 5.6 and 9.0 wt %, respectively) were obtained. The in vitro release studies confirmed much faster release of DOX at pH 5.0 and 6.0 than at pH 7.4. For example, approximately 16, 52, and 61% of drug were released in 22 h, and 23, 83, and 92% of drug were released in 120 h from PEO-g-DOX2.9 at pH 7.4, 6.0 and 5.0, respectively. Notably, confocal laser scanning microscope (CLSM) observations revealed that DOX was released and delivered into the nuclei of RAW 264.7 cells following 24 h of incubation. MTT assays demonstrated that PEO-g-DOX2.9 had pronounced cytotoxic effects to RAW 264.7, HeLa, and 4T1 breast tumor cells with IC50 values of about 26.5, 42.5, and 32.0 mu g DOX equiv/mL, whereas the corresponding polymer carrier PEO-g-hydrazide(4.4) was nontoxic. The In Vivo pharmacokinetics and biodistribution studies in mice showed that PEO-g-DOX2.9 prodrugs had significantly prolonged circulation time and enhanced drug accumulation in the tumor as compared with free DOX We are convinced that endosomal pH-activatable PEO-g-DOX prodrugs have tremendous potential for targeted cancer therapy.