Hepatic steatosis (HS) can exacerbate acute pancreatitis (AP). This study aimed to investigate the relation between α1-antitrypsin (AAT) and acute pancreatitis when patients have HS. Using proteomic profiling, we identified 18 differently expressed proteins pots in the serum of rats with or without HS after surgical establishment of AP. AAT was found to be one of the significantly down-regulated proteins. AAT levels were significantly lower in hepatic steatosis acute pancreatitis (HSAP) than in non-HSAP (NHSAP) (P < 0.001). To explore the clinical significance of these observations, we measured the levels of AAT in the serum of 240 patients with HSAP, NHSAP, fatty liver disease (FLD), or no disease. Compared with healthy controls, serum AAT levels in patients with NHSAP were significantly higher (P < 0.01), while in patients with HSAP serum AAT levels were significantly lower (P < 0.01). Further studies showed that acute physiology and chronic health evaluation (APACHE-II) scores were negatively correlated with serum AAT levels (r = -0.85, P < 0.01). In conclusion, low serum levels of AAT in patients with HSAP are correlated with disease severity and AAT may represent a potential target for therapies aiming to improve pancreatitis.