Abstract Background Neratinib is a tyrosine kinase inhibitor that is used for the therapy of patients with HER2 + breast tumors. However, despite its clinical benefit, resistance to the drug may arise. Here we have created cellular models of neratinib resistance to investigate the mechanisms underlying such resistance. Methods Chronic neratinib exposure of BT474 human HER2 + breast cancer cells resulted in the selection of several clones resistant to the antiproliferative action of the drug. The clones were characterized biochemically and biologically using a variety of techniques. Results These clones retained HER2 levels similar to parental cells. Knockdown experiments showed that the neratinib-resistant clones retained oncogenic dependence on HER2. Moreover, the tyrosine phosphorylation status of BT474 and the resistant clones was equally sensitive to neratinib. Transcriptomic and Western analyses showed that peptidylarginine deiminase 3 was overexpressed in the three neratinib-resistant clones studied but was undetectable in BT474 cells. Experiments performed in the neratinib-resistant clones showed that reduction of PADI3 or inhibition of its function restored sensitivity to the antiproliferative action of neratinib. Moreover, overexpression of FLAG-tagged PADI3 in BT474 cells provoked resistance to the antiproliferative action of neratinib. Conclusions Together, these results uncover a role of PADI3 in the regulation of sensitivity to neratinib in breast cancer cells overexpressing HER2 and open the possibility of using PADI3 inhibitors to fight resistance to neratinib.