Nicotinamide (NAM), the amide form of vitamin B3, is involved in a wide range of biological processes. Recent evidence revealed the anti-inflammatory and anti-oxidant properties of NAM and suggests it may be used as a novel strategy in the prevention of acute liver injury. In the present study, we investigated the potential protective effects of NAM on acetaminophen (APAP)-induced acute liver injury in mice. Mice were treated with NAM at 400mg/kg 30 min before or after administration of APAP at a hepatotoxic dose of 400mg/kg body weight via intraperitoneal injection. Liver injury and the expression of inflammation-related molecules were determined by histological examination and biochemical analysis, respectively. In addition, the survival rate of mice was assessed after APAP administration. Pretreatment with NAM for 30 min significantly decreased plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malondialdehyde (MDA), and diminished histopathologic evidence of hepatic toxicity in mice following APAP administration. Similarly, posttreatment with NAM also decreased plasma ALT and AST levels in APAP-administrated mice. Furthermore, both pretreatment and posttreatment with NAM prolonged the survival rate of acute liver injury mice, accompanied by a significant reduction in the plasma levels of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), and interleukin-6 (IL-6). Together, these findings suggest that NAM possesses protective effects on APAP-induced liver injury, which may involve the anti-inflammatory action.