The study was aimed to investigate the anti-myeloma molecular mechanism of thalidomide (TLD) by detecting gene expression profiles of human myeloma cell line RPMI8226 treated with thalidomide. cDNA microarray were used to detect thousands of gene expression in gene chip. Two cDNA probes were prepared through reverse transcription from mRNA of RPMI8226 cells untreated and treated with TLD. These two probes were labeled with Cy3 and Cy5 fluorescence dyes respectively, then hybridized with cDNA microarray containing 1152 different human genes. The genes with differential expression in RPMI8226 cells treated with TLD for 72 hours were screened by scanning and analysis of computer software, and their functions were explored. The results showed that after co-culture of RPMI 8226 cells with TLD in 100 micromol/L concentration for 72 hours, 22 genes with differential expression were screened. Among these genes, the expressions of 4 genes were down-regulated including rpl32 gene, scya3 gene, mmp1 gene and igbp1 gene. Eighteen genes were up-regulated including wars gene, tubb4q gene, ube1l gene, txnrd1 gene and fyb gene. The study indicated that (1) wars gene encoding tryptophanyl-tRNA synthetase was up-regulated by TLD, while mmp1 gene encoding matrix metalloprotein 1 was down-regulated, they may be related to the inhibition of angiogenesis caused by TLD. (2) scya3 gene encoding macrophage inflammatory protein-1alpha and igbp1 gene encoding immunoglobulin binding protein 1 were down-regulated by TLD, they may play a role in the inhibition of cell proliferation caused by TLD. (3) tubb4q gene encoding tubulin beta4, ube1l gene encoding ubiquitin-activating enzyme E1-like protein and txnrd1 gene encoding thioredoxin reductase 1 were up-regulated by TLD, they may involve in apoptosis of RPMI8226 cells induced by TLD. (4) fyb gene encoding Fyn-binding protein was up regulated by TLD which associated with killing MM cells. It is concluded that 22 differentially expressed genes are involved in protein synthesis and degradation, cell signal transduction, cytoskeletal movement, immune modulation, cell metabolism, regulation of anti-oncogene and cell apoptosis, which relate directly or indirectly to molecular mechanisms of anti-myeloma effects induced by TLD.