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Objective To observe the application effect and mechanism of 5-nitro-2-(3-phenylpropylamino) benzoate acid (NPPB) during the hepatocyte isolation of rats. Methods Twelve Sprague-Dawley rats were randomly divided into two groups,6 in each group: the observation group and the control group, and the hepatocytes of the two groups were isolated by a collagenase perfusion with a modified four-step technique. In addition, we added NPPB to the observation group in the process of using perfusion fluid for digestion. The purity was determined by PAS staining, the hepatocyte yield was detected by cell count technique and the survival rate were determined after 0.4% Trypan blue staining. Cells were cultured in serum-free medium for 24 h, and we collected the supernatant at different times after isolation. LDH release rate content was detected, and the levels of ALB and ALT were detected by automatic biochemistry analyzer. The mRNA level of mitochondrial chloride intracellular channel 4 (CLIC4) was determined by RT-PCR. CLIC4 protein level was detected by Western blotting. Results The hepatocyte yields of the observation group and the control group were (4.41 ±0.20) ×108 and (4.40 ±0.26) ×108 , respectively; the purity was 90%±1% and 89%±2%, respectively; and the survival rates were 88%±2% and 85%±1%, respectively. Significant difference was found between the two groups (all P<0.05). The levels of ALB in the observation group A and the control group were (43.8 ±5.0) and (25.2 ±3.0) g/L, the levels of ALT were (47.8 ±6.0) and (104.3 ±11.7) U/L, and the release rates of LDH were 21.0%±2.7% and 34.0%± 2.4%, respectively; and significant difference was found between the two groups (all P<0.05). The relative expression levels of CLIC4 mRNA in the observation group and the control group were 1.01 ±0.02 and 1.62 ±0.01, the protein expression levels of CLIC4 were 0.38 ±0.02 and 0.52 ±0.03, respectively; and significant difference was found between the two groups (all P<0.05). Conclusions During the hepatocyte isolation, the application of NPPB does not affect the yield, purity and the survival rate and alleviates the membranous structure and the injury of function, which may be related with the inhibition of protein expression of CLIC 4. [ABSTRACT FROM AUTHOR]