Aim: To examine the effect of the mother's heartbeat sound on physiological parameters and pain intensity after blood sampling in neonates in the intensive care unit. Methods: A randomized controlled clinical trial was conducted on 60 neonates admitted to the intensive care unit. They were assigned to intervention and control groups (n = 30 in each group). The intervention group listened to the mother's heartbeat sound, 10 min before up to 10 min after taking arterial blood samples. Pain intensity was measured every 10 min in 3 steps using the neonatal infant pain scale 10 min before the intervention, immediately after and 10 min after the intervention. At the same time, physiologic parameters including oxygen saturation, respiratory rate, heart rate and mean arterial blood pressure were recorded. Data were analyzed using descriptive and inferential statistics using SPSS. Results: Listening to the mother's heartbeat sound did not influence on mean arterial pressure in the intervention group. However, it had significant medium to large effects on oxygen saturation and respiratory rate immediately after and 10 min after the intervention. Also, it had a large effect on heart rate immediately after the intervention (P < 0.05). Also, significant medium to large effects of the intervention on pain intensity immediately after and 10 min after blood sampling were reported (P < 0.05). Conclusion: The mother's heartbeat sound can be used by nurses as a non-pharmacologic and safe intervention along with routine care in order to reduce suffering and pain in neonates undergoing invasive and painful procedures in the intensive care unit.