Background: Heel stick sampling, which is a common procedure in newborns, causes acute pain, and parents are aware of this. Aim: The current study aimed to investigate the effectiveness of maternal-targeted training on newborn pain management, addressing the use of nonpharmacological methods and anxiety. Methods: The study is 2-arm, parallel-group randomized controlled trial. A total of 64 mothers were included in this study. Mothers were randomly allocated to each group; intervention (n:32) and control (n=:32). Training on nonpharmacological pain management in newborns was given to the mothers in the intervention group. Data were collected with an Introductory Information Form, Nonpharmacological Pain Management Use Checklist, and State-Trait Anxiety Inventory. Results: Nonpharmacological methods were used for the procedures in the control group and the intervention group, with an absolute difference of 68.8% between groups. The difference was statistically significant (p < 0.001) and had a large effect (d = −79.222; 95% CI, −9.365 to 670.143. There was no significant difference between the groups in terms of anxiety score (p = 0.558). Conclusion: Demonstrates the clinical relevance and feasibility of training targeting maternal on neonatal pain management during the heel stick sampling procedure. Practice Implications: When non-pharmacological methods in pain management of newborns were recommended by the nurse, maternal involvement in pain management increased significantly, suggesting that nurses have a key role in ensuring parental involvement. Clinical Trial Registration: NCT05173662.