Background Kangaroo care (KC) is an effective technique to prevent injury in newborns due to prematurity and hospitalization. Mothers of preterm newborns experience their own set of physical and mental problems. Such circumstances call for another family member to take care of the newborn. This study compared the effect of KC by mothers and maternal grandmothers on the vital signs of preterm newborns. Methods This parallel randomized controlled trial was done at the neonatal and NICU departments of the hospital in Kuhdasht in Iran. Eighty preterm neonates were selected through convenience sampling according to the eligibility criteria, then by stratified block randomization allocated to two groups. The control group received KC from the mother, and the intervention group received KC from the maternal grandmothers on the vital signs of preterm newborns. Vital signs were assessed 15 min before, during, and after the KC as the primary outcome. The data collection tools included a demographic questionnaire and a form to record the vital signs. Vital signs were measured by a pulse oximeter, an electronic thermometer, and observation. Data were analyzed by the chi-square test, the independent t-test, and the repeated measures ANOVA. Results The vital signs of newborns in each group showed a significant difference before, during, and after receiving KC (P < 0.05). Nevertheless, the vital signs of the newborns did not differ significantly between the mother and the maternal grandmother KC groups (P > 0.05). Conclusion KC by maternal grandmother may stabilize the vital signs of preterm newborns as much as when this type of care is provided by the mother. We, therefore, recommend the provision of KC by the maternal grandmother, as a support and substitute for the mother whenever she is incapable of being at the hospital and to enable the mother to rest. Trial registration Iranian Registry of Clinical Trials IRCT20211225053516N1, March 31, 2022.