Lung cancer is the most common fatal malignancy among all cancers worldwide, and non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. Existing therapies for NSCLC include tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib, which target the epidermal growth factor receptor (EGFR) tyrosine kinase. In this report, we studied the anti-proliferative activity of tanshinone IIA (Tan IIA) from Radix Salvia miltiorrhiza against gefitinib-sensitive and -resistant NSCLC cells. Tan IIA effectively inhibited the growth of both gefitinib-sensitive NSCLC cells harboring the EGFR mutation of exon 19 (PC-9) and gefitinib-resistant NSCLC cells (H1650, H358, AY-01, and A549). We also identified another type of NSCLC cells (PC-14), which were resistant to both TKI and Tan IIA. H1650 and AY-01 harbor an EGFR mutation of exon 19, but are resistant to gefitinib. H358 and A549 harbor a KRAS mutation but no EGFR mutation. Therefore, Tan IIA is a candidate compound for the development of anti-cancer drugs targeting a variety of gefitinib-sensitive and -resistant NSCLC.