The dense extracellular matrix (ECM) in tumor tissue severely hinders the penetration and enrichment of antitumor nanomedicines, which could significantly affect their efficiency. In this study, we used pH-sensitive nanocarriers loaded with collagenase (Col) to remold the tumor microenvironment (TME). Furthermore, we combined the collagenase delivery system with a nanomedicine to improve its penetration and enrichment in the tumor, thereby improving efficacy. We synthesized acetalated dextran (Ace-DEX) with an ideal pH-sensitivity as the carrier material of collagenase. Under mild preparation conditions, collagenase was loaded into Ace-DEX nanoparticles (NPs) with a high loading capacity (>4%) and remained highly active (>90%). Col-carrying NPs (Col-NPs) significantly reduced the tumor collagen content by 15.1%. Pretreatment with Col-NPs increased the accumulation of doxorubicin (DOX)-loaded liposome (DOX-Lipo) in the tumor by 2.8-fold. There were no safety concerns as the Col-NP showed no significant toxicity and reduced Col-induced damage to healthy tissues. Additionally, the number of circulating tumor cells remained unchanged after Col-NP treatment, suggesting no increased risk of tumor metastasis. Because the Col-NP acts essentially independent of the subsequent treatment, it has considerable potential for enhancing many existing delivery systems and drugs for cancer treatment. It may also be used for treating other collagen-related diseases.