Smoking is a negative impact on health. It make the autonomic nervous system imbalance. Therefore, heart rate variability in smokers are lower than non smokers. Objective: This research purpose is to know the differences HRV between smokers and non smokers in 6th and 8th nursing students Universitas Muhammadiyah Yogyakarta. Methodology: This research was non-experimental study with descriptive comparative design and cross sectional approach. This research samples was 40 male students which choosen by purposive sampling techniques. Inclution criteria divided into smokers and non smokers with questionnaire. Heart Rate Variability (HRV) was measured by Electrocardiogram (ECG). The data was analyzed by Mann Whithey. Result: There are significant differences between HRV smokers and non smokers with p = 0.038 (p = < 0.05). HRV smokers lower than non smokers, it was analyzed by Mann Whitney. Conclusion: There are significant differences between HRV smokers and non smokers. HRV smokers lower than non smokers