Background Performing repeated blood cultures after initial positive culture (i.e., follow-up blood cultures [FUBCs]) in patients with gram-negative bacilli (GNB) bacteremia is controversial. We aimed to comprehensively review the association of FUBCs with improvement in patient-relevant clinical outcomes in GNB bacteremia. Methods We performed systematic review and random-effects meta-analysis to calculate summary effect estimates. We used hazard ratios as the effect measure. The primary outcome was 30-day or in-hospital mortality, and secondary outcomes were lengths of treatment and hospital stay. We searched PubMed, Embase, and Cochrane Central Register of Controlled Trials (Central) without language restrictions from inception to April 29, 2022. Original clinical studies evaluating the association between FUBCs and mortality in adult patients with GNB bacteremia were included. FUBC details were reviewed. Two independent reviewers used the Risk of Bias in Non-randomised Studies of Interventions tool. Results We identified nine eligible retrospective studies. In total, 7,778 hospitalized patients with GNB bacteremia were included. The studies were clinically heterogeneous and had a critical risk of bias. The utilization of FUBCs varied across studies (18%–89%). Random-effects meta-analysis of covariate-adjusted estimates found that FUBC use was associated with reduced mortality. Although not a result of the meta-analysis, lengths of treatment and hospital stay were longer for patients with FUBCs than for those without. Adverse events were not reported. Conclusions FUBC acquisitions was associated with lower mortality and longer hospital stay and treatment duration in GNB bacteremia. The risk of bias was critical, and no firm data were available to support mechanisms.