Human amniotic mesenchymal stem cells (hAMSCs) are splendid cell sources for clinical application in the administration of numerous refractory and relapse diseases. Despite the preferable prospect of serum-free (SF) condition for cell product standardization and pathogenic contamination remission, yet the systematic and detailed impact upon hAMSCs at both cellular and transcriptomic levels is largely obscure.For the purpose, we preconditioned hAMSCs under serum-containing (SC) and SF medium for 48 h and compared the biological signatures and biofunctions from the view of cell morphology, immunophenotypes, multi-lineage differentiation in vitro, cell vitality, cytokine expression, and immunosuppressive effect upon the subpopulations of T lymphocytes, together with the PI3K-AKT-mTOR signaling reactivation upon cell vitality. Meanwhile, we took advantage of RNA-SEQ and bioinformatic analyses to verify the gene expression profiling and genetic variation spectrum in the indicated hAMSCs.Compared with those maintained in SC medium, hAMSCs pretreated in SF conditions manifested conservation in cell morphology, immunophenotypes, adipogenic differentiation, and immunosuppressive effect upon the proliferation and activation of most of the T cell subpopulations, but with evaluated cytokine expression (e.g., TGF-β1, IDO1, NOS2) and declined osteogenic differentiation and cell proliferation as well as proapoptotic and apoptotic cells. The declined proliferation in the SF group was efficiently rescued by PI3K-AKT-mTOR signaling reactivation. Notably, hAMSCs cultured in SF and SC conditions revealed similarities in gene expression profiling and variations in genetic mutation at the transcriptome level. Instead, based on the differentially expressed genes and variable shear event analyses, we found those genes were mainly involved in DNA synthesis-, protein metabolism-, and cell vitality-associated biological processes and signaling pathways (e.g., P53, KRAS, PI3K-Akt-mTOR).Collectively, our data revealed the multifaceted cellular and molecular properties of hAMSCs under SC and SF conditions, which suggested the feasibility of serum-free culture for the preferable preparation of standardized cell products for hAMSC drug development and clinical application.© 2022. The Author(s).