The passive targeting via nanomedicine to pancreatic tumor microenvironment (TME) is identified as an optimized therapeutic strategy for pancreatic ductal adenocarcinoma (PDAC) because lacking specific biomarkers and the intractable anatomical position. Herein, an in vitro 3D PDAC model was set up to evaluate the regulation of extracellular matrix (ECM) by an intelligent gemcitabine@nanogel system (GEM@NGH). This GEM@NGH system consisting of a reduction-sensitive core, the payloads of gemcitabine, and the coronal of hyaluronidase arrayed on the cationic surface was fabricated to improve intratumoral penetration and antitumor efficacy. The physicochemical properties, reduction sensitivity, cellular biocompatibility and cytotoxicity, intracellular distribution and therapeutic effects were all evaluated. Particularly, the GEM@NGH system showed excellent ECM eradication and in vitro/vivo solid tumor penetration ability as evaluated by home-built equipment and in vitro 3D PDAC model, which confirmed that GEM@NGH could be disintegrated in the tumoral reductive cytoplasm after internalization and release gemcitabine to exhibit promoted cytotoxicity. In the in vivo therapy, GEM@NGH displayed the highest tumor growth inhibition in PANC-1 tumor-bearing mice with the remarkably increased tumor penetration ability by TME regulation. The results obtained in this study indicate that specifically regulating TME by a well-designed intelligent gemcitabine@nanogel is promising way for the pancreatic cancer therapy.Copyright © 2020. Published by Elsevier B.V.