Gefitinib, the specific inhibitor of the epidermal growth factor receptor (EGFR), may cause growth delay in cancer cell lines. Thorough understanding of the downstream cellular signaling of gefitinib will facilitate the discovery of biomarkers for predicting outcomes and monitoring anti-EGFR therapies, and provide information for key targets for therapeutic intervention. In this study, we investigated the role of transducer of erbB2.1 (TOB1) in gefitinib therapy. Using the lung carcinoma cell lines A549 and NCI-H1975, the results suggested that gefitinib might mediate cell cycle arrest in lung cancer cells at least by targeting TOB1 expression. Gefitinib treatment caused cell cycle arrest predominantly at the G1 phase, which is associated with TOB1 nuclear translocation and its interaction with cyclin D1. We also showed that knockdown of TOB1 expression by RNAi rescued lung cancer cells from gefitinib-induced cell-proliferative arrest. These results suggest that TOB1 interaction with cyclin D1 and nuclear translocation is directly involved in the gefitinib-induced anti-proliferative cell cycle arrest.