Objective: To assess organizational culture in neonatal intensive care units (NICUs) in Japan. Design: Cross-sectional survey of organizational culture. Setting: Forty NICUs across Japan. Participants: Physicians and nurses who worked in NICUs (n = 2006). Main Outcome Measures: The Competing Values Framework (CVF) was used to assess the organizational culture of the study population. The 20-item CVF was divided into four culture archetypes: Group, Developmental, Hierarchical and Rational. We calculated geometric means (gmean) and 95% bootstrap confidence intervals of the individual dimensions by unit and occupation. The median number of staff, beds, physicians' work hours and work engagement were also calculated to examine the differences by culture archetypes. Results: Group (gmean = 34.6) and Hierarchical (gmean = 31.7) culture archetypes were higher than Developmental (gmean = 16.3) and Rational (gmean = 17.4) among physicians as a whole. Hierarchical (gmean = 36.3) was the highest followed by Group (gmean = 25.8), Developmental (gmean = 16.3) and Rational (gmean = 21.7) among nurses as a whole. Units with dominant Hierarchical culture had a slightly higher number of physicians (median = 7) than dominant Group culture (median = 6). Units with dominant Group culture had a higher number of beds (median = 12) than dominant Hierarchical culture (median = 9) among physicians. Nurses from units with a dominant Group culture (median = 2.8) had slightly higher work engagement compared with those in units with a dominant Hierarchical culture (median = 2.6). Conclusions: Our findings revealed that organizational culture in NICUs varies depending on occupation and group size. Group and Hierarchical cultures predominated in Japanese NICUs. Assessing organizational culture will provide insights into the perceptions of unit values to improve quality of care.