Butyrate inhibits the proliferation of cancer cells, but the early molecular events initiated by butyrate have not been fully identified. Herein, butyrate is shown to affect the growth arrest and DNA damage-inducible gene 153 (GADD153) in HCT-116 human colon adenocarcinoma cells. Despite absence of any detectable cellular DNA damage, the expression of GADD153 was upregulated before several features characteristic of apoptosis appeared. Butyrate-induced upregulation of GADD153 mRNA was attenuated by actinomycin D, but apparently not by cycloheximide. In investigating possible involvement of MAPK in mediating the effect of butyrate on GADD153 mRNA expression, the extracellular regulated kinase (ERK) inhibitor PD98059, but neither the JNK inhibitor SP600125 nor the p38 MAPK inhibitor SB203580, blunted the ability of butyrate to upregulate GADD153 mRNA expression. U0126, a selective inhibitor of upstream MEK, had a similar effect as PD98059 on butyrate-induced GADD153 mRNA upregulation. Collectively, these findings suggest that butyrate caused activation of the GADD153 gene at the level of transcription involving mainly the MEK/ERK branch of the MAPK signal transduction pathway. Moreover, these molecular events were not the result of any DNA damage and occurred before several features characteristic of apoptosis became evident.