Background: Data on the pathogens responsible for nosocomial bloodstream infection (BSI) and their antimicrobial resistance (AMR) in Hubei province are limited. This study was conducted to determine the major pathogens causing BSI and to characterize their AMR. Methods: Data from the China Antimicrobial Resistance Surveillance System (CARSS) from 2014 to 2016 were analyzed retrospectively. Results: Escherichia coil, Staphylococcus aureus and Klebsiella pneumoniae were the most common pathogens responsible for nosocomial BSI. Individuals aged 0-5 years and >= 40 years old were the major demographics at risk of infection by E. coli, K pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter cloacae, while individuals aged 0-5 years were the major demographic at risk of infection by S. aureus, Enterococcus faecalis, E. faecium, Streptococcus pneumoniae and Stenotrophomonas maltophilia. The frequencies of E. coli and K. pneumoniae isolates resistant to cefotaxime were 59.1% and 24.3%, respectively, and the frequencies of resistant isolates to ceftazidime were 42.9% and 27.2%, respectively. From 2014 to 2016, the frequency of extended-spectrum beta-lactamase (ESBL)-positive E. coli declined from 29.07 to 24.5%, and the frequency of ESBL-positive K. pneumoniae declined from 18.64 to 12.33%. The frequency of carbapenem-resistant (CR) E. coli was below 0.5%, but 1-10% of K. pneumoniae isolates were CR. Conclusions: The emergence of methicillin-resistant S. aureus and the expansion of ESBL and fluoroquinolone resistance among Gram-negative Enterobacteriaceae increased AMR severity. Carbapenemase-producing K pneumoniae isolates responsible for nosocomial BSI increased year over year and effective infection control measures should be taken to prevent them from spreading.