For evaluation of cell therapy strategies using human mesenchymal stem cells (hMSCs), it is important to be able to trace transplanted cells and their distribution in tissues, for example, cartilage, over time. The aim of the study was to determine effects on cell viability, traceability, and chondrogenic differentiation of hMSCs after iron labeling with iron sucrose. hMSCs were collected (seven donors, 13-57 years) from patients undergoing spinal surgery. Two subsets of experiments were performed. (1) Iron labeling of hMSCs: 1 mg/mL of Venofer(®) (iron sucrose) was added (16 h) to cultures. hMSCs were examined for uptake of iron sucrose (Prussian blue staining) and cell viability (flow cytometry). (2) Iron-labeled hMSCs (passage 4) (n=4, pellet mass), 200,000 cells/tube, were cultured (DMEM-HG) with 10 ng/mL TGFβ and compared with controls (from each donor). The pellets were harvested at days 7, 14, and 28. Real-time PCR, IHC, and histology were used to evaluate SOX9, ACAN, C6S, and COL2A1 expression. Mean number of cells containing iron deposits was 98.1% and mean cell viability was 92.7% (no significant difference compared with unlabeled control cells). Pellets containing iron-labeled cells expressed COL2A1 on protein level (all time points), in similar levels as controls, and glycosaminoglycan accumulation was observed in iron-labeled pellets (day 14 or day 28). Results were supported by the expression of chondrogenic genes SOX9, ACAN, and COL2A1. The results in vitro indicate that iron sucrose can be used as a cell tracer for evaluation of cellular distribution in vivo after transplantation of MSCs and thus contribute with important knowledge when exploring new treatment strategies for degenerated cartilaginous tissues.