BackgroundCosmc (C1GalT1C1) mutation could cause aberrantO-glycosylation and result in expression of Tn antigen on the surface of tumor cells (Tn+cells), which is associated with the metastasis and prognosis of cancer progression. Mesenchymal stem cells (MSCs) could participate in immunoregulation, tissue damage repair, and tumor inhibition and be seen as an ideal candidate for tumor therapy due to their inherent capacity to migrate to tumor sites. However, their therapeutic effectiveness in different tumors is inconsistent and still controversial. Of note, emerging data reveal that side population (SP) cells have a stronger multilineage developmental potential than main population cells and can function as stem/progenitor cells. The effect of SP cells derived from MSCs on the biological behaviors and theO-glycosylation status of tumor cells remains unclear.MethodsSP cells were isolated from human umbilical cord MSCs (hUCMSCs) and human placenta MSCs (hPMSCs). Tn+cells (LS174T-Tn+and HT-29-Tn+cells) and matching Tn−cells (LS174T-Tn−and HT-29-Tn−cells) were isolated from human colorectal cancer cell (CRC) lines LS174T and HT-29 by immune magnetic beads. The proliferation, migration, apoptosis, Tn antigen expression, andO-glycome in Tn+and Tn−CRC cells before and after co-cultured with SP-MSCs were detected using real-time cell Analysis (RTCA), flow cytometry (FCM), and cellularO-glycome reporter/amplification (CORA), respectively. Cosmc protein andO-glycosyltransferase (T-synthase and C3GnT) activity in CRC cells were, respectively, assessed using western blotting and fluorescence method.ResultsBoth SP cells derived from hUCMSCs and hPMSCs could inhibit proliferation and migration, promote apoptosis of CRC cells, significantly reduce Tn antigen expression on Tn+CRC cells, generate new core 1-, 2-, and 3-derivedO-glycans, increase T-synthase and C3GnT activity, and elevate the levels of Cosmc and T-synthase protein.ConclusionSP-hUCMSCs and SP-hPMSCs could inhibit proliferation and migration and promote apoptosis of Tn+CRC cells via increasingO-glycosyltransferase activity to modifyO-glycosylation status, which further adds a new dimension to the treatment of CRC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-023-03334-3.