This study investigates the impact of replicative senescence on human mesenchymal stem cells (MSCs). MSCs, which are promising for cellular therapy, undergo morphological changes, reduced expression of surface markers, and arrested proliferation after 43 to 77 days of cultivation. Adipogenic differentiation potential decreases, while osteogenic differentiation potential increases. mRNA expression profiling reveals a consistent pattern of alterations in global gene expression, with genes involved in cell cycle, DNA replication, and repair being significantly down-regulated. Genes from chromosome 4q21 are over-represented among differentially regulated transcripts. MiRNA expression profiling shows up-regulation of hsa-miR-371, hsa-miR-369-3P, hsa-miR-29c, hsa-miR-499, and hsa-let-7f. These findings indicate that replicative senescence of MSCs is a continuous and organized process, affecting phenotype, differentiation potential, gene expression, and miRNA profiles. These changes need to be considered for the therapeutic application of MSC preparations.This study investigates the impact of replicative senescence on human mesenchymal stem cells (MSCs). MSCs, which are promising for cellular therapy, undergo morphological changes, reduced expression of surface markers, and arrested proliferation after 43 to 77 days of cultivation. Adipogenic differentiation potential decreases, while osteogenic differentiation potential increases. mRNA expression profiling reveals a consistent pattern of alterations in global gene expression, with genes involved in cell cycle, DNA replication, and repair being significantly down-regulated. Genes from chromosome 4q21 are over-represented among differentially regulated transcripts. MiRNA expression profiling shows up-regulation of hsa-miR-371, hsa-miR-369-3P, hsa-miR-29c, hsa-miR-499, and hsa-let-7f. These findings indicate that replicative senescence of MSCs is a continuous and organized process, affecting phenotype, differentiation potential, gene expression, and miRNA profiles. These changes need to be considered for the therapeutic application of MSC preparations.