Diallyl disulfide (DADS), a sulfur compound derived from garlic, has been shown to have protective effects against colon carcinogenesis in several studies performed in rodent models. However, its molecular mechanism of action remains unclear. This study was designed to confirm the anti-proliferative activity of DADS and to screen for differentially expressed genes induced by DADS in human colon cancer cells with the aim of exploring its possible anticancer mechanisms. The anti-proliferative capability of DADS in the HT-29 human colon cancer cells was analyzed by MTT assays and flow cytometry. The differences in gene expression between DADS-treated (experimental group) and untreated (control group) HT-29 cells were identified using two-directional suppression subtractive hybridization (SSH). Semi-quantitative reverse transcription polymerase chain reaction (semi-RT-PCR) was selected to confirm the results obtained by SSH. Based on the results, a dose- and time-dependent growth inhibition was observed in the DADS-treated HT-29 cells. Forty-nine known genes and a new gene were found to be involved in the anti-proliferative effects of DADS by SSH analysis, and two cDNA libraries, DHDG and DHUG, containing both up- and down-regulated genes in colon tumor cells, were constructed. These genes were related to transduction, cell proliferation/growth/apoptosis and secreted/extracellular matrix proteins. Semi-RT-PCR results showed an expression pattern consistent with that of the SSH analysis. In conclusion, DADS showed anti-proliferative effects on colon cancer HT-29 cells, and DHDG and DHUG genes were found to be involved in this process. Further studies on the identification and description of these genes may allow a better understanding of the protective roles of DADS in colon carcinogenesis.