Background: Shortages of essential medicines, a long-standing issue in healthcare, apply equally to antimicrobial agents, a group of essential drugs necessary for sustainable healthcare. The WHO categorized essential medicines into the access, watch, and reserve groups. Older antimicrobials, in particular, were categorized into the access group, meaning that these drugs are in theory widely available at an affordable cost. The shortage of essential antimicrobial agents like cefazolin leads to increased consumption of alternative antimicrobial agents with broad-spectrum activity, which often has the undesirable consequence of defeating antimicrobial stewardship efforts in inpatient settings and potentially promoting antimicrobial resistance. In Japan, cefazolin has been in critically short supply since March 2019. Cefazolin is a first-line agent against common infectious diseases and in surgical antimicrobial prophylaxis, and its shortage has substantially impacted inpatient care. The aim of the present study was to investigate changes in antimicrobial practice at a tertiary-care center in Japan following the emergence of the national cefazolin shortage in March 2019. Methods: Data on each antimicrobial use are logged as days of therapy (DOT) per 1,000 patient days (PD) for antimicrobial stewardship purposes at the study institution. We extracted weekly data from September 2018 to September 2019 to evaluate the impact of the national cefazolin shortage on antimicrobial use at our tertiary-care center. Changes in weekly antimicrobial use and the weekly incidence of Clostridium difficile infections were analyzed by interrupted time series analysis. We also investigated changes in antimicrobial practice at selected situations. Results: As weekly cefazolin use significantly declined after the emergence of the national shortage, use of third-generation cephalosporin (+18.9 DOT per 1,000 PD for intercept [P < .001] and +0.65 DOT per 1,000 PD per week for trend [P = .037]) and clindamycin (18 DOT per 1,000 PD for intercept [P = .008] and 0.12 DOT per 1,000 PD per week for trend [P = .003]) significantly increased. Significant changes in antimicrobial practice were also observed in surgical antimicrobial prophylaxis: third-generation cephalosporin use increased from 1.0% (31 of 3,032) to 62.9% (2,237 of 3,554) (P < .001). However, no significant change in the incidence of Clostridium difficile infection was observed during the study period: +1.72 per 10,000 PD for intercept (P = .12) and 0.12 per 10,000 PD per week for the trend (P = .09). Conclusions: The national cefazolin shortage had a significantly negative impact on patient care and led to increased use of alternative, broader-spectrum antimicrobials, which are not ideal choices either for prophylaxis or treatment.