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Introduction: Using non-invasive techniques are necessary to reduce the premature infant pain. This study aimed to determine the effect of Kangaroo Mother Care (KMC) on premature newborns’ pain due to invasive procedures in neonatal intensive care unit. Materials & Methods: In a quasi-experimental study, 70 hospitalized preterm infants were selected and then randomly divided in two case and control groups. In case group, the physiological and behavioral signs of pain 15 minutes after KMC (minutes zero), during the invasive painful procedures (minutes 15) and 15 minutes after the re-establishment of KMC (minutes 30) and in control group, that signs in three same time zero (15 minutes after supine position of Infant in the incubator), 15 (during a painful procedure) and 30 (15 minutes after painful procedures) was observed and recorded by physiological & behavioral sign scale. Findings: According to results, there were significant differences between case and control group in heart rate, oxygen saturation, facial flushing and sweating from physiological signs (P<0/05). There were significant differences between case and control group in breathing pattern, the level of consciousness (P<0/05), positioning arms and legs (P<0/01) from behavioral signs. Discussion & Conclusion: KMC is an applicable and non-invasive method to relieve hospitalized premature newborns’ pain due to painful invasive procedures in neonatal intensive care unit.