The present study provides supportive evidence for the effective prevention and treatment of lipopolysaccharide (LPS)-induced hepatocyte injury in neonatal mice by N-acetylcysteine (NAC). Hepatocytes of neonatal mice were obtained through collagenase digestion of the liver. The hepatocytes were treated either with LPS (10 ?g/mL) alone or with NAC (5 mmol/L) for 1 h before the addition of LPS (10 ?g/mL). After LPS treatment, 12 wells of the cultured hepatocytes and supernatants were harvested at 0, 6, and 12 h, respectively. Levels of alanine aminotransferase (ALT) and nitric oxide (NO) in the supernatant were biochemically quantified and reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect the expression of inducible nitric oxide synthase (iNOS) mRNA after different treatments. At 0 h following the treatment of primary cultured hepatocytes with LPS, the levels of ALT, NO and iNOS mRNA in the supernatant were (21.1 ± 4.78) u/L, (1.6 ± 0.31) ?mol/L and 0.17 ± 0.023, respectively; at 6 h, (59.8 ± 8.59) u/L, (6.6 ± 0.81) ?mol/L, and 0.71 ± 0.091; and at 12 h, (89.6 ± 15.30) u/L, (7.8 ± 1.01) ?mol/L, and 0.71 ± 0.097. The levels of ALT, NO and iNOS mRNA at 6 and 12 h increased significantly, compared to those at 0 h (P < 0.01). In contrast to LPS treatment alone, pretreatment with NAC before LPS addition significantly reduced the levels of ALT, NO and iNOS mRNA in the supernatant at 6 h to (40.8 ± 7.30) u/L, (3.2 ± 0.71) ?mol/L, and 0.41 ± 0.060; and at 12 h to (55.4 ± 5.48) u/L, (4.0 ± 0.71) ?mol/L, and 0.40 ± 0.067, respectively (P < 0.01). However, the levels of ALT, NO and iNOS mRNA at 0 h did not change significantly with both treatment approaches. NAC has protective effects in hepatocytes of neonatal mice against LPS-induced injury as shown by the reduced levels of ALT, NO and iNOS mRNA when primary hepatocytes were treated with NAC prior to LPS stimulation. We postulate that NAC exhibits its protective function by inhibiting LPS-induced transcription of iNOS, resulting in decreased levels of NO.