Non‑small cell lung cancer (NSCLC) remains the leading cause of cancer‑related mortality despite the fact that great advances have been made in therapeutic treatment methods. Therefore, in the present study, the role of adenovirus-mediated retinoblastoma 94 (Ad‑RB94) gene therapy in NSCLC was investigated. Following treatment with Ad‑RB94, the proportion of A549 cells in the G2/M phase was increased. In the mouse xenograft model, the overexpression of RB94 inhibited the tumor growth compared with the control group and the Ad-‑LacZ-treated group. In the transplanted tumors, the overexpression of RB94 induced the apoptosis of tumors as well as an increase in the mRNA levels of cyclinB1. In conclusion, the results of the present study suggested that RB94 may effectively inhibit NSCLC tumor cell growth by inducing G2/M cell cycle arrest and apoptosis, indicating that RB94 may be a promising candidate for adjuvant therapy with radiation or chemotherapy in NSCLC.