Here, a targeted delivery system was developed based on silk fibroin nanoparticles (SFNPs) for the systemic delivery of gemcitabine (Gem) to treat induced lung tumor in a mice model. For targeting the tumorigenic lung tissue, SP5-52 peptide was conjugated to Gem-loaded SFNPs. Different methods were used to characterize the structural and physicochemical properties of the SFNPs. The prepared nanoparticles (NPs) showed suitable characteristics in terms of size, zeta potential, morphology, and structural properties. Moreover, the targeted Gem-loaded SFNPs showed higher cytotoxicity, cellular uptake, and accumulation in the lung tissue in comparison to the nontargeted SFNPs and control groups. Afterward, a mice model with induced lung tumor was developed by intratracheal injection of Lewis lung carcinoma (LL/2) cells into the lungs for assessing the therapeutic efficacy of the prepared drug delivery system. The histopathological assessments and single-photon-emission computed tomography-CT radiographs showed successful lung tumor induction. Moreover, the obtained results showed higher potential of targeted Gem-loaded SFNPs in treating induced lung tumor compared with that of the control groups. Higher survival rate, less mortality, and no sign of metastasis were also observed in those animals treated with targeted NPs based on the histological and radiological analyses. This study presented an effective anticancer drug delivery system for specific targeting of induced lung tumor that could be useful in treating malignant lung cancers in future.