Objective To analyze the distribution and antimicrobial resistance of pathogens isolated from patients with acute pancreatitis (AP) and infection, and guide rational use of antimicrobial agents in clinic. Methods Pathogens and antimicrobial resistance of patients with AP and infection from 2016 to 2018 were retrospectively analyzed, AP patients were divided into severe acute pancreatitis (SAP) group and non-SAP group according to the severity of disease, distribution of pathogens and isolation of multidrug-resistant organisms (MDROs) were compared between two groups. Results 262 patients with AP and infection were enrolled in study, 144 (54.96%) of whom were with mixed infection. A total of 291 strains of pathogens were isolated, 207 (71.13%) of which were gram-negative strains and 84 (28.87%) were gram-positive strains; the top three pathogens were Escherichia coli (n=71), Klebsiella pneumoniae (n=45) and Acinetobacter baumannii (n=40). Acinetobacter baumannii infection rate and MDRO infection rate in SAP group were higher than those in non-SAP group (P<0.05). Biliary AP patients were more susceptible to MDRO infection than non-biliary AP patients (P<0.05). Resistance rates of Escherichia coli and Klebsiella pneumoniae to the third generation cephalosporins and fluoroquinolones were all over 50%, and to imipenem were 9.86% and 35.56% respectively; resistance rates of Acinetobacter baumannii to ampicillin/sulbactam and amikacin were both<35%,but to other antimicrobial agents were all >60%; no gram-positive strains were found to be resistant to linezolid, vancomycin, tegacycline and teicoplanin. Conclusion Gram-negative bacteria are the main pathogen causing infection in AP patients, which are highly drug resistant and multi-resistant, MDRO infection poses a serious threat to the treatment of SAP patients.