Polymer-drug conjugates has significantly improved the anti-tumor efficacy of chemotherapeutic drugs and alleviated their side effects. N-(1,3-dihydroxypropan-2-yl) methacrylamide (DHPMA) copolymer was synthesized via RAFT polymerization and polymer-doxorubicin (DOX) (diblock pDHPMA-DOX) were formed by conjugation, resulting in a self-aggregation-induced nanoprodrug with a favorable size of 21 nm and great stability. The nanoprodrug with a molecular weight (MW) of 95 kDa released drugs in response to tumor microenvironmental pH variations and they were enzymatically hydrolyzed into low MW segments (45 kDa). The nanoprodrug was transported through the endolysosomal pathway, released the drug into the cytoplasm and some was localized in the mitochondria, resulting in disruption of the cellular actin cytoskeleton. Cellular apoptosis was also associated with reduction in the mitochondrial potential caused by the nanoprodrug. Notably, the nanoprodrug had a significantly prolonged blood circulation time with an elimination half time of 9.8 h, displayed high accumulation within tumors, and improved the in vivo therapeutic efficacy against 4T1 xenograft tumors compared to free DOX. The tumor xenograft immunohistochemistry study clearly indicated tumor inhibition was through the inhibition of cell proliferation and antiangiogenic effects. Our studies demonstrated that the diblock pDHPMA-DOX nanoprodrug with a controlled molecular structure is promising to alleviate adverse effects of free DOX and have a great potential as an efficient anticancer agent. STATEMENT OF SIGNIFICANCE: In this work, we prepared a biodegradable diblock DHPMA polymer-doxorubicin conjugate via one-pot of RAFT polymerization and conjugate chemistry. The conjugate-based nanoprodrug was internalized by endocytosis to intracellularly release DOX and further induce disruption of mitochondrial functions, actin cytoskeleton alterations and cellular apoptosis. The nanoprodrug with a high molecular weight (MW) (95 kDa) showed a long blood circulation time and achieved high accumulation into tumors. The nanoprodrug was degraded into low MW (∼45 kDa) products below the renal threshold, which ensured its biosafety. Additionally, the multi-stimuli-responsive nanoprodrug demonstrated an enhanced antitumor efficacy against 4T1 breast tumors and alleviated side effects, showing a great potential as an efficient and safe anticancer agent.