Non-suicidal self-injury (NSSI) involves the deliberate harm of one's body without the intent to commit suicide and is particularly concerning among adolescents. Teachers play a critical role as gatekeepers in identifying and addressing self-harm, underscoring the importance of their knowledge and response strategies in this area. This study explored how teachers' knowledge, attitudes towards NSSI, perceived roles, and workplace barriers affect their responses to students exhibiting NSSI behaviors. A cross-sectional survey was conducted among 203 middle and high school teachers in Israel. Data were collected during July and August 2023 using six validated questionnaires. Higher levels of teacher knowledge, positive attitudes, and strong role perceptions correlated with more effective responses to NSSI, whereas increased workplace barriers tended to diminish response efficacy. Positive correlations emerged between role perception and both knowledge and attitudes, whereas negative correlations emerged between workplace barriers, attitudes, and role perceptions. Teaching experience moderated the impact of role perception and workplace barriers on responses. Significant differences were observed between regular and special education settings, although no differences were noted in referral rates or years of seniority. These findings suggest that enhancing teacher knowledge and attitudes towards NSSI, while addressing workplace barriers, can improve response efficacy in educational settings.