Currently, there is no effective treatment for liver fibrosis. In vitro studies on the activation of hepatic stellate cells (HSCs), which is responsible for liver fibrosis, have been used as a drug screening system for it. However, even if some drug shows an inhibitory effect on HSC activation, its anti-fibrotic effect is required to be confirmed in liver fibrosis animal models, which further requires a lot of time and cost. In the present study, we tried to establish an ex vivo model of liver fibrosis using precision-cut liver slices (PCLSs) to solve such problems. PCLSs of 250 µm thickness were prepared from male C57BL/6J mice using a vibratome and cultured in RPMI medium in a 5% CO₂ incubator. Although cellular ATP content was decreased on day 1 compared to day 0, it was then maintained until day 5, suggesting that the ex vivo model is viable for at least 5 days. Treatment with Et-OH (50, 100 mM), one of the liver injury stimuli, for 5 days increased mRNA expression of Acta2 and Col1a1, liver fibrosis markers, in PCLSs. DIF-1 (50, 100 µM), which has an anti-fibrotic effect, significantly suppressed the Et-OH-induced increases in the markers. These results suggest that the ex vivo model using PCLSs is useful as a drug screening system for the development of drugs for treatment of liver fibrosis.