Increasing the molecular weight of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers by using micellar structures could result in more pronounced enhanced permeability and retention effect, thus increase the tumor accumulation of drug. However, most micellar formulations are relatively unstable and release their drug non-specifically. To improve on these disadvantages, we developed a micellar drug delivery system based on self-assembly of HPMA copolymers. Amphiphilic conjugates were synthesized by conjugating the hydrophobic drug doxorubicin and hydrophobic β-sitosterol to the hydrophilic HPMA polymer backbone via pH-sensitive hydrazone linkages. This linkage is quite stable at physiological pH but hydrolyzes easily at acidic pH. After conjugates self-assembly into micelles, HPMA copolymer side chains were cross-linked through the hydrazone linkages to ensure micelle stability in the blood. Using this approach, cross-linked micelles were obtained with molecular weight of 1030 KD and diameter of 10-20 nm. These micelles remained stable with undetectable doxorubicin release at pH 7.4 or mouse plasma, whereas collapsed quickly with 80% of the drug released at pH 5 which corresponds to the pH of lyso/endosome compartments of tumor cells. Both cross-linked and non-cross-linked micelles displayed similar in vitro anti-tumor activity as linear copolymer conjugates in Hep G2 and A549 cancer cell lines with internalization mechanism by caveolin, clathrin, and giant macropinocytosis. In vivo studies in an H22 mouse xenograft model of hepatocarcinoma showed the tumor accumulation (1633 μCi/Lh) and anti-tumor rate (71.8%) of cross-linked micelles were significantly higher than non-cross-linked ones (698 μCi/Lh, 64.3%). Neither type of micelle showed significant toxicity in heart, lung, liver, spleen or kidney. These results suggest that cross-linked HPMA copolymer micelles with pH-sensitivity and biodegradability show excellent potential as carriers of anti-cancer drugs.