To study the genetic basis of spontaneous transformation of mesenchymal stem cells (MSCs) and clinical application value MSCs were isolated by combining utilization of density gradient centrifugation and adherence screening. After cell homogeneity analysis by flow cytometry, spontaneous transformation MSCs were isolated after six-month in vitro expansion. Then cell total RNA was obtained with Trizol reagent and studied for gene expression profile. Differentially expressed genes between normal MSCs and spontaneous transformation MSCs in cDNA microarray were determined by real-time RT-PCR for validation of the microarray data. Forty-four genes were differentially expressed after spontaneous transformation of MSCs, among which 21 were up-regulated and 23 down-regulated. The result of real-time RT-PCR was in accordance with that of the cDNA microarray. Several genes in SHH, Notch, TGFβ/BMPs signal pathway play an important role in spontaneous transformation of MSCs.