Background and objective: Diabetes Mellitus (DM) is not only a health issue but also an economic issue in India. Incorrect insulin injection techniques can lead to side effects such as pain, lipohypertrophy, and poor glycemic control. We designed the present study to assess nurses’ knowledge about safe insulin administration and evaluate the role of a planned teaching programme on knowledge and practices of safe insulin injection techniques in a group of nurses in tertiary care hospital.Methods: This is a pre-post design to study the effectiveness of the structured training programme - one hour of didactic lecture followed by demonstration of safe injection practices. Demographic data and knowledge about safe insulin practices were collected at baseline. We conducted two post training assessments–day one and three months after training. The injection practices were assessed using a check-list. We used the random effects linear regression model to identify factors associated with change in scores over these three observations.Results: The mean (SD) scores for insulin knowledge at baseline was 6.81 (2.28). It significantly increased to 16.85 (1.84) immediately after training (p < .001). These scores reduced significantly after three months compared with post-training scores (14.18 [2.14]; p < .001). A significantly higher proportion of nurses had used re-suspension technique for insulin injection after three months (76.3% vs 52.5%, p = .003) and cleaned the injection site with alcohol swab before injection (93.8% vs. 75.0%, p = .001). On an average, knowledge scores changed by -0.15 (95% CI: -0.29, -0.02; p = .03) with each unit increase in age (years). The average score in nurses with a degree was significantly higher compared with those who had a diploma (1.02, 95% CI: 0.28, 1.76; p = .007).Conclusions: The study demonstrated that insulin injection practices improve with adequate guidance and information. However, there is a need to have a regular training programme to sustain the practices. Certain practices such as site rotation and assessing lipo-hypertrophy, and the relation between these two should be emphasized in these sessions.