Objective To evaluate whether warm povidone-iodine (PI) application before peripherally inserted central catheter (PICC) placement eased pain related to the procedure in premature infants and reduced the duration of the procedure and the number of attempts. Methods A prospective randomized controlled trial was conducted with infants born before 32 weeks of gestation who required the first placement of the PICC. Skin disinfection was performed with warm PI before the procedure in the warm PI(W-PI) group, whereas PI kept at room temperature was used in the regular PI(R-PI) group. NPASS scores of the infants were evaluated three times: at baseline(T0), during skin preparation(T1), and during needle insertion(T2). Results Fifty-two infants (26 in the W-PI group,26 in the R-PI group) were enrolled in the study. The perinatal and baseline demographic characteristics did not significantly differ between the two groups. While the median NPASS scores at T0 and T2 were similar between the groups, the median T1 score was significantly higher in the R-PI group(p = .019). While the median NPASS scores at T1 and T2 were similar in the R-PI group, there was a significant difference in the W-PI group, with NPASS scores being significantly lower at T1 compared to T2. The results demonstrate that skin disinfection was just as painful as needle insertion in the R-PI group. The duration of the procedure and the number of needle insertions were significantly lower in the W-PI group. Conclusions Before invasive interventions, such as PICC insertion, we recommend warm PI as a part of non-pharmacological pain management.