ObjectiveTo evaluate the distribution and antimicrobial resistance of pathogens in neonates. MethodsBlood culture specimens from hospitalized neonates in the neonatal intensive care unit (NICU) of a maternal and child heath hospital between January 1, 2013 and December 31, 2015 were investigated retrospectively, blood culture and antimicrobial susceptibility testing results were analyzed with WHONET 5.6 software.ResultsA total of 255 strains were isolated from blood culture specimens, including 219 strains（85.88%） of gramnegative bacteria, 29 strains（11.37%） of grampositive bacteria, and 7 strains（2.75%） of fungi. The isolated bacteria were mainly Klebsiella pneumoniae (n=157), Escherichia coli (n=35), and Streptococcus B (n=13). Resistance of Escherichia coli to ampicillin and piperacillin were 97.14% and 94.29% respectively, all were sensitive to cefoxitin, imipenem, meropenem, ertapenem, and amikacin. Resistance rates of Klebsiella pneumoniae to piperacillin, cefazolin, and cefuroxime were 93.63%, 87.90%, and 85.35% respectively, but was sensitive to amikacin. Resistance rates of the main gramnegative bacteria to enzyme inhibitor (amoxicillin/clavulanic acid, ticarcillin/clavulanic acid), cephalosporins (ceftazidime, cefoxitin), and sulfonamides (sulfamethoxazole compound) increased, difference were all statistically significant (all P<0.01).ConclusionKlebsiella pneumoniae and Escherichia coli are the major pathogens causing bloodstream infection in NICU, and resistance rates to a multiple antimicrobial agents are high, empirical medication for antiinfection treatment should be combined with local data.