High cofilin-1 levels have been shown to be an accurate prognostic biomarker in non-small cell lung cancer (NSCLC) and a predictive factor in drug resistance. Herein we explore the role of cofilin-1 in cis-diamminedichloroplatinum(II) (cisplatin) resistance. We evaluated cofilin-1 levels in intrinsically cisplatin-resistant A549 (ICR-A549) cells and determined the cisplatin toxicity in A549 cells transiently transfected and overexpressing CFL1 plasmid. Moreover, expression levels (activity) of the CFL1 gene network were analyzed in a cisplatin-resistant human lung adenocarcinoma cell panel. ICR-A549 cells, selected by challenging parental cells with 10-fold drug GI50 value, presented a sixfold increase in cisplatin GI50 value and an increased cofilin-1 immunocontent (P < 0.01). In addition, cells transfected with cofilin-1 became more resistant to cisplatin (P < 0.01). High activity of the CFL1 gene network was found in a cisplatin-resistant adenocarcinoma cell panel (P < 0.01). In vitro evidences suggest that cofilin-1 is a biological predictor of cisplatin resistance, supporting new treatment initiatives based on cofilin-1 levels to guide chemotherapeutic interventions in NSCLC patients.