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Background: Universal Zero-Markup Drug Policy (UZMDP) mandates no price mark-ups on any drug dispensed by a healthcare institution, and covers the medicines not included in the China’s National Essential Medicine System. Five tertiary hospitals in Beijing, China implemented UZMDP in 2012. Its impacts on these hospitals are unknown. We described the effects of UZMDP on a participating hospital, Jishuitan Hospital, Beijing, China (JST). Methods: This retrospective longitudinal study examined the hospital-level data of JST and city-level data of tertiary hospitals of Beijing, China (BJT) 2009–2015. Rank-sum tests and join-point regression analyses were used to assess absolute changes and differences in trends, respectively. Results: In absolute terms, after the UZDMP implementation, there were increased annual patient-visits and decreased ratios of medicine-to-healthcare-charges (RMOH) in JST outpatient and inpatient services; however, in outpatient service, physician work-days decreased and physician-workload and inflation-adjusted per-visit healthcare charges increased, while the inpatient physician work-days increased and inpatient mortality-rate reduced. Interestingly, the decreasing trend in inpatient mortality-rate was neutralized after UZDMP implementation. Compared with BJT and under influence of UZDMP, JST outpatient and inpatient services both had increasing trends in annual patient-visits (annual percentage changes[APC] = 8.1% and 6.5%, respectively) and decreasing trends in RMOH (APC = -4.3% and -5.4%, respectively), while JST outpatient services had increasing trend in inflation-adjusted per-visit healthcare charges (APC = 3.4%) and JST inpatient service had decreasing trend in inflation-adjusted per-visit medicine-charges (APC = -5.2%). Conclusion: Implementation of UZMDP seems to increase annual patient-visits, reduce RMOH and have different impacts on outpatient and inpatient services in a Chinese urban tertiary hospital. [ABSTRACT FROM AUTHOR]