Citosol (thiamylal sodium) is one of generally used anesthetic-sedative agents for clinical patients, and it has not been reported to show induction of cytotoxic effects in cancer cells, especially in mice leukemia RAW 264.7 cells in vitro. In the present study, we investigated the cytotoxic effects of citosol on mice leukemic RAW 264.7 cells, including the effects on protein and gene expression levels which are determined by Western blotting and DNA microarray methods, respectively. Results indicated that citosol induced cell morphological changes, cytotoxic effect, and induction of apoptosis in RAW 264.7 cells. Western blotting analysis demonstrated that citosol promoted the levels of Fas, cytochrome c, caspase 9 and 3 active form and Bax levels, but it suppressed Bcl-xl protein level that may lead to apoptotic death in RAW 264.7 cells. Furthermore, DNA microarray assay indicated that citosol significantly promoted the expression of 5 genes (Gm4884, Gm10883, Lce1c, Lrg1, and LOC100045878) and significantly inhibited the expression of 24 genes (Gm10679, Zfp617, LOC621831, Gm5929, Snord116, Gm3994, LOC380994, Gm5592, LOC380994, LOC280487, Gm4638, Tex24, A530064D06Rik, BC094916, EG668725, Gm189, Hist2h3c2, Gm8020, Snord115, Gm3079, Olfr198, Tdh, Snord115, and Olfr1249). Based on these observations, citosol induced cell apoptosis and influenced gene expression in mice leukemia RAW 264.7 cells in vitro.