Mesenchymal stem cells (MSCs) are considered to be critical for regenerative medicine. However, long-term culturing of MSCs will induce aging of MSCs, and thereafter impair cellular function. Changes in proteomics have been reported to be involved in cell aging, and therefore investigations on cell aging of MSCs at levels of proteomics and post-translational protein modifications (PTM) are ultimately important. In the present study, human umbilical cord mesenchymal stem cells (hUMSCs) were exposed to different culture conditions for either 7 or 30 days. Proteins changes during cell culture were investigated using tandem mass tag (TMT) labeling quantitative approach, and N-glycosylation patterns were analyzed using multistage mass spectrometry. We identified 66 proteins (fold change >1.50) that were differentially expressed in long-term culture. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that aging exerts a side effect on hUMSCs by affecting various molecular functions and biological processes, such as lysosome, autophagy and post-translational protein modification. Glycosylation analysis indicates that cell N-glycan patterns are associated with aging of MSCs. Our results presented here should contribute to future studies on cell aging and cellular quality controls related to MSCs as regenerative medicine.Copyright © 2022. Published by Elsevier Inc.