Painful and/or damaged nipples associated with breastfeeding are common and represent a challenge for both the persons experiencing nipple pain and/or trauma and for those providing treatment. However, evidence-based data has been insufficient to demonstrably minimize these common reasons for failure to initiate or continue successful breastfeeding. The aim of this study was to evaluate the efficacy of specific-grade highly purified anhydrous (HPA) lanolin versus expressed breastmilk (EBM) for the treatment of painful and damaged nipples associated with breastfeeding in a prospective controlled clinical trial evaluating 84 lactating mothers. Nipple trauma and healing rates were rated by the Nipple Trauma Score. Nipple pain intensity was assessed on a visual analog scale. Outcome parameters were in favor of the HPA lanolin group, reaching statistical significance for healing rates, nipple trauma and nipple pain. In our study, we found HPA lanolin more effective than EBM, inducing faster healing of nipple trauma (absolute risk reduction of 0.43) and reducing nipple pain (absolute risk reduction of 0.61 on day 3). We concluded that HPA lanolin, combined with breastfeeding education, was more effective than EBM, combined with breastfeeding education, in reducing nipple pain and promoting healing of nipple trauma.