Background: The role of follow up blood cultures (FUBC) in the management of gram-negative bloodstream infection (GN-BSI) remains controversial. This retrospective cohort study examines the association between obtaining FUBC and mortality in GN-BSI. Methods: Hospitalized adults with community-onset GN-BSI at Prisma Health-Midlands hospitals in South Carolina, USA from January 1, 2010 to June 30, 2015 were identified. Patients who died or were discharged from hospital within 72 h were excluded to minimize impact of survival and selection biases on results, respectively. Multivariate Cox proportional hazards regression was used to examine association between obtaining FUBC and 28-day all-cause mortality after adjustment for the propensity to obtain FUBC. Findings: Among 766 patients with GN-BSI, 219 (28.6%) had FUBC obtained and 15 of 219 (6.8%) FUBC were persistently positive. Overall, median age was 67 years, 438 (57%) were women, 457 (60%) had urinary source of infection, and 426 (56%) had BSI due to Escherichia coli. Mortality was significantly lower in patients who had FUBC obtained than in those who did not have FUBC (6.3% vs. 11.7%, log-rank p = 0.03). Obtaining FUBC was independently associated with reduced mortality (hazards ratio 0.47, 95% confidence intervals: 0.23À0.87; p = 0.02) after adjustments for age, chronic comorbidities, acute severity of illness, appropriateness of empirical antimicrobial therapy, and propensity to obtain FUBC. Interpretation: Improved survival in hospitalized patients with GN-BSI who had FUBC is consistent with the results of recent publications from Italy and North Carolina supporting utilization of FUBC in management of GN-BSI. Funding: This study had no funding source.