The human amniotic membrane (hAM) is a promising therapeutic source in regenerative medicine due to its rich content of growth factors and bioactive molecules. Its clinical application, however, has remained limited by challenges in controlled delivery and compositional complexity. To address these issues, we developed a nanoparticle-based formulation (NF-hAM) to encapsulate hAM proteins and enhance their targeted delivery to mesenchymal stem cells (MSCs) and fibroblasts. NF-hAM was synthesized using soy lecithin and chitosan, forming spherical nanoparticles with well-defined morphology. Physicochemical characterization confirmed successful protein encapsulation and nanoparticle stability. studies demonstrated that NF-hAM was biocompatible and significantly promoted the proliferation of bone marrow-derived MSCs (BMSCs), adipose-derived MSCs (AMSCs), and human skin fibroblasts (HSFs). In HSFs, NF-hAM also induced cytoskeletal remodeling, suggesting enhanced cellular activity and functional responsiveness. Additionally, NF-hAM enhanced the osteogenic and adipogenic differentiation potential of BMSCs. Metabolic analyses further revealed that NF-hAM stimulated glycolytic activity, a key metabolic pathway associated with self-renewal and proliferation. Our findings highlight the potential of NF-hAM as a therapeutic nanomedicine for regenerative medical applications.