Background: Planning the number of registered nurses (RN) per shift in the neonatal intensive care unit (NICU) is a constant stressor and overtime is often used to assure adequate nurse to patient ratios at high costs. Aim: To identify the factors associated with shift-to-shift variations in the use of RN overtime in the NICU and assess the economic impacts of reducing overtime. Methods: We developed a two-year retrospective study in a NICU (CHU de Québec, Level 3 unit, capacity of 51 beds). Detailed administrative data for each shift of the day (night, day, evening) was collected. Non-modifiable organizational factors included patient volume, patient acuity, number of admissions, season, days of the week and work shift. The modifiable factors included the paid hours not at the bedside and the implementation of a bundle to reduce RN overtime (increase in full-time nurse positions and conversion of 10% of RN to regular 12-hour shifts). Multivariate linear regression models were used to assess the association between organizational factors and RN overtime per shift. Results: A total of 2184 shifts were included. Mean RN overtime per shift was 9.5±10.4 h corresponding to 4.7±5.2% of total hours worked per shift. RN overtime use was influenced by non-modifiable factors including unit occupancy, season and the number of acute patients. Paid hours not at the bedside were associated with overtime. Also, the implementation of a bundle to reduce RN overtime brought the mean RN overtime from 11.7±11.2 h to 6.5±8.5 h (p<0.001). This was associated with a reduction in nursing costs per patient day [386±10 $ vs. 381±7 $ (p<0.001)]. That corresponded to a yearly 102,948$ cost reduction. Conclusion: Reducing RN overtime in the NICU is associated with cost reduction.