Cross-sectional studies of hospital-level administrative data have suggested that 4 nurse staffing practices-using adequate staffing levels, higher proportions of registered nurses (RNs) (skill mix), and more educated and experienced RNs-are each associated with reduced hospital mortality. To increase the validity of this evidence, patient-level longitudinal studies assessing the simultaneous associations of these staffing practices with mortality are required. A dynamic cohort of 146,349 adult medical, surgical, and intensive care patients admitted to a Canadian University Health Center was followed for 7 years (2010-2017). We used a multivariable Cox proportional hazards model to estimate the associations between patients' time-varying cumulative exposure to measures of RN understaffing, skill mix, education, and experience, each relative to nursing unit and shift means, and the hazard of in-hospital mortality, while adjusting for patient and nursing unit characteristics, and modeling the current nursing unit of hospitalization as a random effect. Overall, 4854 in-hospital deaths occurred during 3,478,603 patient-shifts of follow-up (13.95 deaths/10,000 patient-shifts). In multivariable analyses, every 5% increase in the cumulative proportion of understaffed shifts was associated with a 1.0% increase in mortality (hazard ratio: 1.010; 95% confidence interval: 1.002-1.017; P=0.009). Moreover, every 5% increase in the cumulative proportion of worked hours by baccalaureate-prepared RNs was associated with a 2.0% reduction of mortality (hazard ratio: 0.980; 95% confidence interval: 0.965-0.995, P=0.008). RN experience and skill mix were not significantly associated with mortality. Reducing the frequency of understaffed shifts and increasing the proportion of baccalaureate-prepared RNs are associated with reduced hospital mortality.