Introduction: To evaluate insulin injection knowledge, attitudes, and practices of nurses across China in order to provide reference for the formulation of a national unified standard of insulin injection practice and the targeted implementation of standardized training on insulin injection for nurses.Methods: We enrolled nurses who worked and injected insulin at grassroot hospitals including community health service centers and township clinics, secondary and tertiary care hospitals across China between July 28, 2019 and August 30, 2019. A nurse insulin injection knowledge, attitude, and practice questionnaire was used to evaluate the knowledge, attitude, and practice level of nurses. Results: A total of 223,368 nurses were included in the study. The mean knowledge score was 13.70 ± 3.30 and 35.19% had a poor knowledge score. The mean attitude score was 17.18 ± 2.69 for the study nurses; merely 3.15% had a poor attitude score. The mean practice score of the study population was 83.03 ± 8.16 Xinjuan Wu and Fang Zhao contributed equally to this work.