We performed a population-based study to examine the influence of healthcare-associated acquisition on pathogen distribution, antimicrobial resistance, short- and long-term mortality of community-onset Gram-negative bloodstream infections (BSI). We identified 733 unique patients with community-onset Gram-negative BSI (306 healthcare-associated and 427 community-acquired) among Olmsted County, Minnesota, residents from 1 January 1998 to 31 December 2007. Multivariate logistic regression was used to examine the association between healthcare-associated acquisition and microbiological etiology and antimicrobial resistance. Multivariate Cox proportional hazards regression was used to evaluate the influence of the site of acquisition on mortality. Healthcare-associated acquisition was predictive of Pseudomonas aeruginosa (odds ratio [OR] 3.14, 95% confidence intervals [CI]: 1.59-6.57) and the group of Enterobacter, Citrobacter, and Serratia species (OR 2.23, 95% CI: 1.21-4.21) as causative pathogens of community-onset Gram-negative BSI. Healthcare-associated acquisition was also predictive of fluoroquinolone resistance among community-onset Gram-negative bloodstream isolates (OR 2.27, 95% CI: 1.18-4.53). Healthcare-associated acquisition of BSI was independently associated with higher 28-day (hazard ratio [HR] 3.73, 95% CI: 2.13-6.93) and 1-year mortality (HR 3.60, 95% CI: 2.57-5.15). Because of differences in pathogen distribution, antimicrobial resistance, and outcomes between healthcare-associated and community-acquired Gram-negative BSI, identification of patients with healthcare-associated acquisition of BSI is essential to optimize empiric antimicrobial therapy.