Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSCs transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray (SAM) identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and anti-apoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to loose their stem cell identity as demonstrated by osteogenic, chondrogenic and adipogenic differentiation assays with both transduced and wild type MSCs, although certain degree of alterations occurred. Histologic analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cell while enabling non-invasive imaging in living animals to study the biodistribution and other biological activities of the cells.