Preterm infants are exposed to serious health problems that require advanced highly specialized nursing skills in order to sustain their life. Developmental positioning is one of an essential skill for neonatal intensive care unit nurses. This study aimed to investigate the effectiveness of developmentally supportive positioning on preterm infants' pain responses at NICU. A Quasi experimental design was used to conduct the study at neonatal intensive care unit on a sample of (56)preterm infants who admitted through nine months and were equally divided randomly into the study and control group after fulfilling the inclusion criteria using demographic characteristics of Preterm infants. Infant Position Assessment Tool (IPAT) and Preterm Infant Pain Profile (PIPP) Tool. The results indicated that all the preterm infants (100%) had an unacceptable positioning in both groups, and none of preterm infants in both groups had no-or-minimal pain at baseline assessment. While after one week of intervention about two third (64.3%) of the infants were placed in an acceptable position in the study group and only less than one quarter (21.4%) of the preterm infants in the control group were placed in an acceptable position. Regarding pain response at day seven of intervention five minutes after morning routine care, about half (53.6%) of infants in the study group had no-or-minimal pain while none of infants in the control group had no-or-minimal pain and about one third (32.1%) of infants in the control group had severe pain and none of the infants in the study group had severe pain. The present study concluded that preterm infants who were placed in developmentally supportive positioning had acceptable position and exhibited less pain scores. It is recommended to replicate the present study on a larger sample.