The mechanism of ethanol-induced hepatotoxicity was complicated, accompanied by the over-expressions of the cytochrome P450 2E1 (CYP2E1), heat shock protein 70 (Hsp70) and the nuclear factor specificity protein 1 (SP1). Kaempferol (Kaem) could protect the ethanol-induced hepatotoxicity likely by inhibiting the CYP2E1 expression and activity. This study investigated the protective mechanism(s) of kaempferol on ethanol-induced toxicity by dynamic alteration of SP1, Hsp70 and CYP2E1 among the nucleus and different organelles in hepatocytes. After ethanol treatment alone and co-incubation hepatocytes with kaempferol, protein levels of CYP2E1, Hsp70, and SP1 were determined in vitro (western blotting and immunofluorescence). Hepatocytes' viability was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods. Glutathione (GSH) levels were evaluated for ethanol-induced oxidative stress. In the ethanol-treated hepatocytes, kaempferol decreased protein levels of CYP2E1 in both microsome and mitochondria, cytosolic Hsp70 and SP1 in nuclear and cytosol, and the oxidative stress and increased the cell viability compared to those of ethanol group. Collectively, our findings propose that the protective mechanism of kaempferol is involved in the synchronous, early and persistent inhibitions of mitochondrial and microsomal CYP2E1, cytosolic Hsp70 and nuclear and cytosolic SP1 in mouse primary hepatocytes' injury induced by ethanol.