Objective:To investigate the distribution and resistance of pathogens isolated from blood culture of patients in the First Affiliated Hospital of Nanjing Medical University in 2014. Methods: All blood samples were cultured by BACTEC FX. Automatic detection machine of VITEK-2 Compact was used for identification of bacteria and fungus, as well as the susceptibility of non-fastidious bacteria. Susceptibility of streptococcus was tested by K-B method while susceptibility of fungus was tested by ATB FUNGUS 3. Whonet 5.6 software was used for statistical analysis. Results: The total of pathogen strains isolated from blood culture in 2014 was 691. The positive rates of three kinds of blood culture model, bilateral double bottles, bilateral single bottles and unilateral single bottle, were 12.3%, 11.4% and 9.4%, respectively. The top 5 bacteria were Escherichia coli (22.3%), coagulase negative staphylococcus (CNS, 17.5%), Klebsiella pneumonia (11.0%), Streptococcus (7.2%) and enterococcus (7.1%). The separation rate of gram-negative bacteria (50.4%) was higher than that of gram-positive bacteria (41.0%). The pathogens of blood culture were mainly isolated from the department of hematology, intensive care unit, general surgery, and infection. The resistance rates of Escherichia coli to cephalosporins, monobactam, quinolones were 44.2%-79.9%, and the resistance rates to aminoglycosides, cephamycins, β-lactamase/β-lactamase inhibitors, and carbapenems were less than 25.0%. Enterococcus had a higher than 50.0% resistance rate to many kinds of antibiotics. We also found that the resistance rates of Acinetobacter baumanmii to all the clinical antibiotics were higher than 60.0%. Conclusion: Multiple sets of inspection of blood culture helped to improve the positive rate. The pathogenic bacteria were dominated by gram-negative bacteria which had a relatively higher resistance rate to clinical antibiotics.