In this study, a novel array electrospinning collector was devised to generate two distinct regenerated silk fibroin (SF) fibrous membranes: ordered and disordered. Leveraging electrostatic forces during the electrospinning process allowed precise control over the orientation of SF fiber, resulting in the creation of the membranes comprising both aligned and randomly arranged fiber layers. This innovative approach resulted in the development of large-area membranes featuring exceptional stability due to their alternating patterned structure, achievable through expansion using the collector. The study delved into exploring the potential of these membranes in augmenting wound healing efficiency. Conducting in vitro toxicity assays with Adipose Tissue-Derived Mesenchymal Stem Cells (AD-MSCs) and Normal Human Dermal Fibroblasts (NHDFs) confirmed the biocompatibility of the SF membranes. Notably, AD-MSCs exhibited the ability to discern distinct microenvironments established by electrospun SF membranes, translating this recognition into a conditioned medium (CM). Evaluations focused on the paracrine effects of AD-MSCs in promoting migration via CM, drawing comparisons with secretions from NHDFs. The study employed two distinct strategies: firstly, the utilization of paracrine secretion by AD-MSCs to encourages cellular migration, particularly on SF flat membranes where their secretion enhanced cellular migration while elevating the protein concentration of the CMs. Secondly, the exploitation of physical cues from SF electrospun membranes to guide and augment cell recruitment, thereby enhancing wound healing. Observations centered on monitoring cell migration and documenting the influence of SF materials on inducing morphological changes in both AD-MSCs and NHDFs., The ordered membrane, in particular, exhibited pronounced effectiveness in guiding directional cell migration. This research underscores the revelation that customizable microenvironments facilitated by SF membranes optimize the paracrine products of MSCs and offer valuable physical cues, presenting novel prospects for enhancing wound healing efficiency.