Impairment of extracellular matrix (ECM) remodeling is observed in the tumor microenvironment or fibrosis and results in excessive collagen production and/or decreased degradation by metalloproteinases MMPs. Thanks to their local application and transient effects, physical stimuli appear as attractive tools to remodel ECM. We assessed the potential of pulsed electric field technology, classically applied to drug delivery, to induce collagen remodeling at the tissue scale. A sophisticated in vitro tissue-engineered human dermal substitute was used to demonstrate that microsecond and millisecond pulsed electric fields induced 1) a rapid modulation (4h after electrostimulation) of mRNA’s genes composing the matrisome, particularly a down-regulation of pro-collagens and ECM maturation’s enzymes such as transglutaminase TG2 and LOX-like; 2) a transient decrease in pro-collagens production and hydroxyproline tissue content within a week after electrostimulation; 3) a long-lasting ROS-dependent over-activation of MMPs for at least 48h and 4) a down-regulation of TGF-β. These observations underpin that pulsed electric fields, a technology already approved for clinical use combined with anti-cancer agents, are particularly promising to provide local and effective treatment of abnormal ECM.