Background: The incidence of pain in ill infants hospitalized in the neonatal ward is unavoidable.. The aim of the present study was to compare the effects of two methods of breastfeeding and oral glucose 10% on the severity of venipuncture-induced pain and physiological indicators in infants hospitalized in the neonatal ward of Seyyed Al-Shohada Hospital in Kerman in 2019.Methods: The present study is a clinical trial study with three groups and with a pre-test and a post-test. It was conducted on 120 neonates who met the inclusion criteria. Infants were selected by a convenient sampling method, and they were randomly divided into three groups. The study instruments included a neonatal demographic questionnaire, and neonatal infant pain scale and a checklist for recording physiological indicators. Then, the data were analyzed using SPSS25 software.Results: The results of the study showed that the mean score of pain severity before the intervention was the same and zero in the three groups (P > 0.05), but during venipuncture and after that, a statistically significant difference was observed among three groups (P < 0.05). The highest mean pain during venipuncture was observed in the control group (86.95) and the lowest mean pain was observed in the breastfeeding intervention group (35.91). After venipuncture, the lowest mean pain was observed in the breastfeeding intervention group (40.40). Also, there was no significant difference between the mean physiological indicators (temperature and heart rate) in different stages of the venipuncture (before, during, and after) in three groups (P > 0.05).Conclusion: Based on the results of the study, breastfeeding is more effective than oral glucose 10% in reducing the severity of infant pain during venipuncture. Breastfeeding is an easy and inexpensive and available method that can be easily implemented with proper training for the mother and it can be used as a method of reducing infant pain by nurses in neonatal intensive care units to ensure the normal growth and development of the infant and prevent the physical effects and discomfort of infants.