Background and Aim: Given that suicide represents a major psychiatric emergency and the age of suicide attempts is declining, early identification and timely intervention are crucial for prevention. This study aimed to evaluate the effectiveness of a gatekeeper training intervention on suicide prevention knowledge, attitudes, and behaviors among healthcare workers in Sirjan, Iran. Materials and Methods: In this clinical trialconducted in 2025, 40 healthcare workers in Sirjan were selected via purposive sampling and randomly allocated into experimental and control groups (n=20). The experimental group participated in a gatekeeper training program consisting of seven 90-minute sessions over two months, while the control group received no intervention. Data were collected using the Literacy of Suicide Scale (LOSS), the Gatekeeper Behavior Scale (GBS), and the Attitudes Towards Suicide (ATTS) questionnaire. Statistical analysis was performed using descriptive statistics, independent samples t-test, chi-square test, and two-way repeated-measures ANOVA in SPSS (version 24). Results: At baseline, there were no statistically significant differences between the experimental and control groups regarding knowledge, attitude, and behavior scores (p>0.05). However, ANOVA revealed significant main and interaction effects across the three assessment time points (pre-test, post-test, and follow-up) (p<0.001), indicating a substantial improvement in suicide prevention knowledge, attitudes, and behaviors in the experimental group over time compared to the control group. Conclusion: The gatekeeper training intervention effectively enhanced suicide prevention knowledge, attitudes, and behaviors among healthcare workers.Therefore, integrating this educational program into healthcare settings is recommended to facilitate the early detection, support, and referral of individuals at risk of suicide.