OBJECTIVE: To evaluate the impact of implementing a clinical care guideline for uncomplicated gram-negative bloodstream infections (GN-BSI) within a health system. DESIGN: Retrospective, quasi-experimental study. SETTING: A large academic safety-net institution. PARTICIPANTS: Adults (≥18 years) with GN-BSI, defined by at least one positive blood culture for specific gram-negative organisms. Patients with polymicrobial cultures or contaminants were excluded. INTERVENTIONS: Implementation of a GN-BSI clinical care guideline based on a 2021 consensus statement, emphasizing 7-day antibiotic courses, use of highly bioavailable oral antibiotics, and minimizing repeat blood cultures. RESULTS: The study included 147 patients pre-intervention and 169 post-intervention. Interrupted time series analysis showed a reduction in the median duration of therapy (-2.3 days, CONCLUSIONS: Guideline implementation was associated with shorter antibiotic therapy durations, increased use of guideline-concordant oral antibiotics, and fewer repeat blood cultures without compromising patient outcomes. These findings support the effectiveness of institutional guidelines in standardizing care, optimizing resource utilization, and promoting evidence-based practices in infectious disease management.