Abstract： Objectives Construct a subtractive library of Caski cell line induced by exposing to the space environment by suppression subtractive hybridization and pave the way to explain the molecular mechanisms of the changes at the gene level. Methods Super SMART cDNA synthesis and suppression subtractive hybridization (SSH) were performed to isolate differentially expressed cDNA fragments from strains subclonal 48A9 cell line. cDNA from the 48A9 cell line were used as " tester" , and the other from the control Caski cell line as "driver". Subtractive products were directly inserted into T/A cloning vector, and then transformed into host bacteria to set up a subtractive cDNA library of specially or highly expressed genes in strains subclonal 48A9 cell line. Results mRNA were directly extracted and purified with good quality. Double strand cDNA were reverse transcripted integratedly, and then cut by Rsa I into even length short segments. Liga-tion was identified as high effective. After two hybridizations, a subtractive library of differentially expressed genes in strains subclonal 48A9 cell line was successfully constructed by SSH. Conclusion SSH is an effective approach to isolate differentially expressed genes.