Population-based studies of Gram-negative bloodstream infection (BSI) in children are lacking. Therefore, we performed this population-based investigation in Olmsted County, Minnesota, to determine the incidence rate, site of acquisition, and outcome of Gram-negative BSI in children aged ⩽18 years. We used Kaplan-Meier method and Cox proportional hazard regression for mortality analysis. We identified 56 unique children with Gram-negative BSI during the past decade. The gender-adjusted incidence rate of Gram-negative BSI per 100 000 person-years was 129·7 [95% confidence interval (CI) 77·8-181·6]) in infants, with a sharp decline to 14·6 (95% CI 6·0-23·2) and 7·6 (95% CI 4·3-10·9) in children aged 1-4 and 5-18 years, respectively. The urinary tract was the most commonly identified source of infection (34%) and Escherichia coli was the most common pathogen isolated (38%). Over two-thirds (68%) of children had underlying medical conditions that predisposed to Gram-negative BSI. The overall 28-day and 1-year all-cause mortality rates were 11% (95% CI 3-18) and 18% (95% CI 8-28), respectively. Younger age and number of underlying medical conditions were associated with 28-day and 1-year mortality, respectively. Nosocomial or healthcare-associated acquisition was associated with both 28-day and 1-year mortality.