PURPOSE: This study was conducted to assess the effects of lights-out at nighttime on body weight, physiological variables, and behavioral status in premature infants and to provide basic data for applying lights-out at night time in premature infants. METHODS: Premature infants of over 32 weeks' corrected age were included in this study (January 2015-June 2015), and were allocated to two groups according to the lights-out at night for 5 hours: study group and control group. Lights-out was applied to the study group from midnight for five hours in a quiet environment. RESULTS: Fifty-two infants were included in the study: 26 in the study group and 26 in the control group. Growth rates of body weight, height, and head circumference were higher in the study group compared to the control group, but there were no statistical differences. In the physiological variables, heart rate decreased by 6.9 beats per minute in the study group, but it increased by 2.7 beats per minute in the control group (P<0.0001) during applied 5 hours at night. Anderson Behavioral State Score decreased in the study group compared to the control group (P=0.042). CONCLUSION: Lights-out at night decreased the heart rate and made the behavioral status more stable. To understand the effects of lights-out on long-term growth and development of premature infants at the highest risk of delayed growth and development, further studies with a larger number of premature infants are needed.