Objective To investigate and analyze the gap between the current clinical practice and guidelines of insulin injection knowledge,attitude and practice,and to provide a basis for further construction of an intervention program for insulin injection knowledge,attitude and practice that conform to the actual situation in China. Methods The stratified random convenience sampling method was used to investigate 5550 nurses from 3 primary,7 secondary and 11 tertiary hospitals from July 28 to August 30,2019,using the general information questionnaire and the questionnaire of nurses’ knowledge,attitude and practice on insulin injection.SPSS26.0 was used for independent sample T test,variance analysis and correlation analysis on the influencing factors of knowledge,attitude and practice,and Mplus8.3 was used to analyze the path of influencing factors of knowledge,attitude and practice. Results The scores of nurse knowledge, attitude and practice on insulin injection were 68.17±15.76,87.22±13.34 and 93.74±7.97,respectively. There was a positive correlation between nurses’ insulin injection knowledge and attitude,knowledge and practice,attitude and practice scores(r=0.321,0.298,0.561,P<0.001). Gender and professional title were the main influencing factors of nurses’ insulin injection knowledge scores. Knowledge,hospital level,number of beds,professional title,department,and the last time you engaged in insulin injection were the main influencing factors of nurses’ attitude score of insulin injection. Hospital level,education,knowledge and belief were the main influencing factors of nurses’ insulin injection practice score. Attitude played a partially mediating role in knowledge and nurses’ insulin injection practice. The total effect value was 0.299;the indirect effect was 0.163;the intermediary effect accounted for 54.5% of the total effect value. Conclusion There is still a big gap between clinical practice and guidelines of nurse knowledge,attitude and practice on insulin injection. Nursing managers should strengthen the evaluation of hospital nurses’ knowledge,attitude and practice of insulin injection,and take intervention measures according to the characteristics and influencing factors of nurses’ knowledge,attitude and practice of insulin injection to improve their knowledge,attitude and practice.