Background: Kangaroo care helps reduce pain in newborns, especially those associated with a variety of procedures that are part of routine medical and nursing care for premature infants. Aim: This study aimed to evaluate the effect of kangaroo care on pain response in premature infants during blood sampling. Research design: A quasi-experimental study design was used to achieve the aim of this study. Setting: This study was conducted in a neonatal intensive care unit. Sample: The study included a purposive sample of premature infants, which were divided randomly into two groups (45 in each group).Tool: one tool was used; 1st part; Characteristics of premature infants and 2nd part; Premature Infant Pain Profile (PIPP). Results: Studied premature infants' total score of pain reflect significant differences compared to a control group. Kangaroo care was effective in reducing pain scores in premature infants before, during, and after blood sampling, with significantly reducing pain severity in premature infants compared to the control group. Conclusion: Premature infants who received Kangaroo care exhibited less pain scores during blood sampling compared to premature infants who did not receive it. Recommendations: Kangaroo care should be used as a part of the routine care to reduce pain during painful procedures in premature infants admitted in neonatal intensive care unit.