Background: Neonatal period is one of the most important critical phases of humanlife. Intensive care unit has a stressful environment for the infant in which the patient will be under the pressure of factors such as noise, nursing intervention and harsh light; the most important factor in this regard being separation from parents. Objectives: This study aimed to investigate the effect of skin-to-skin care by fathers on salivary cortisol of his infant. Methods: This study was a randomized clinical trial on 45 premature infants paired by their fathers at the neonatal intensive care unit (NICU) of Tabriz Alzahra teaching hospital conducted during November 2015. The control group received standard care and the intervention group had 45 minutes of skin to skin care. Saliva samples were collected from infants before, during, and after this intervention to measure the cortisol level. The SPSS 13 statistical software was used to analyze the data with the significance level of P < 0.05. Results: Salivary cortisol in babies in the control group had a mean value of 66.36 (SD = 71.22) and intervention group a mean value of 59.56 (SD = 59.20) (P = 0.56). Conclusions: Both groups showed decreasing cortisol levels during the study, the reduction in the skin-to-skin care group was more than the control group, but with no significant difference. Thus, making it possible for fathers to take care of their infants in an effective, helpful and secure way.