ObjectiveTo analyze the species and antimicrobial resistance of pathogens from blood culture of hospitalized patients in a tertiary firstclass hospital, and provide reference for clinical rational antimicrobial use. MethodsData of pathogens isolated from blood culture of patients in a hospital between January 2015 and November 2017 were collected, distribution, antimicrobial resistance characteristics, and positive alarming time were studied retrospectively. ResultsA total of 7 153 blood culture specimens were obtained, 655 strains of pathogens were isolated. 370 strains (56.49%) were gramnegative bacilli, 262 (40.00%) were grampositive cocci, and 23(3.51%) were fungi. The top 5 pathogens were Escherichia coli, coagulase negative staphylococcus (Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis), Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. The average positive alarming time of blood culture from early to late were Enterobacteriaceae(15.50 h), Staphylococcus spp. (18.00 h), nonfermentative bacteria (20.30 h), Enterococcus spp. (22.50 h), Streptococcus spp. (24.15 h), and fungi (38.35 h). Department of hematologyoncology（24.89%） had the highest isolation rate of pathogens from blood culture. Grampositive cocci was found not to be resistant to linezolid, teicoplanin, and vancomycin; imipenem and meropenem had good antimicrobial activity against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii (resistance rates were all<5%); resistance rates of Acinetobacter baumannii to piperacillin, ceftazidine, cefepime, gentamicin, and compound sulfamethoxazole were all >65%. ConclusionAntimicrobial resistance of hospitalized patients with bloodstream infection in this hospital is serious and distribution in various clinical departments is different. Clinicians should standardize the use of antimicrobial agents according to antimicrobial susceptibility testing results of positive pathogens from blood culture.