Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide. The use of a combination of chemotherapy drugs and zinc oxide nanoparticles (ZnO-NPs), which have proven to induce tumor-selective cell death, reduce the drug resistance and reduce the side effects in vitro. In the present study, we developed ZnO-NPs loaded with both cisplatin (Cp) and gemcitabine (Gem) (ZnO-NPs(Cp/Gem)), then the morphologies and the size distribution of ZnO-NPs(Cp/Gem) particles were observed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Also, MTT, western blot and Annexin V-PI were used to assess the anti-tumor role of ZnO-NPs(Cp/Gem) in A549 cells. The viability for A549 cells showed a significant decrease in the ZnO NPs(Cp/Gem) group, respectively relative to Cp, Gem, the combination of Cp and Gem (Cp+Gem), and ZnO-NPs loaded with Cp (ZnO-NPs(Cp)) or Gem (ZnO-NPs(Gem)). Furthermore, ZnO-NPs(Cp/Gem) remarkably enhanced the apoptosis-promoting effect of Cp and Gem in A549 cells. The xenograft model showed that Zno-NPS (Cp/Gem) significantly enhanced the inhibition of Cp and Gem on tumor formation. The above results suggested that therapy of NSCLC with ZnO-NPs(Cp/Gem) could enhance the cytotoxic action of chemotherapeutic agents synergistically, indicating a promising potential for ZnO-NPs in antitumor applications.