Dense and stiff extracellular matrix (ECM) in heterogeneous tumor tissues can inhibit deep penetration of nanoparticle drug carriers and decreases their therapeutic efficacy. Herein, we suggest the ECM remodeling strategy by the pulsed high intensity focused ultrasound (Pulsed-HIFU) technology for enhanced tumor-targeting of nanoparticles. First, we clearly observed that the tumor-targeting efficacy and tissue penetration of intravenously injected Cy5.5-labled glycol chitosan nanoparticles (Cy5.5-CNPs) were greatly inhibited in tumor tissue containing high collagen and hyaluronan contents in ECM-rich A549 tumor-bearing mice, compared to in ECM-less SCC7. When collagenase or hyaluronidase was treated by intra-tumoral injection, the amount of collagen and hyaluronan decreased in ECM-rich A549 tumor tissues and more Cy5.5-CNPs penetrated inside the tumor tissue, confirmed using non-invasive optical imaging. Finally, in order to break down the stiff ECM structure, ECM-rich A549 tumor tissues were treated with the relatively low power of Pulse-HIFU (20 W/cm2), wherein acute tissue damage was not observed. As we expected, the A549 tumor tissues showed the remodeling of ECM structure after non-invasive Pulsed-HIFU exposure, which resulted in the increased blood flow, decreased collagen contents, and enhanced penetration of CNPS. Importantly, the tumor targeting efficiency in Pulsed-HIFU-treated A549 tumor tissues was 2.5 times higher than that of untreated tumor tissues. These overall results demonstrate that ECM remodeling and disruption of collagen structure by Pulse-HIFU is promising strategy to enhance the deep penetration and enhanced tumor targeting of nanoparticles in ECM-rich tumor tissues.