Folic acid-conjugated temozolomide (TMZ)-loaded chitosan nanoparticles (CS-TMZ-FLA-NP) were developed to target lung cancer in the anticipation that folic acid would increase the affinity of nanoparticles for cancer cells. CS-TMZ-FLA-NP showed the highest anti-proliferative effect on the lung cancer cells in comparison to free TMZ and CS-TMZ-NP (nanoparticles without folic acid). A cellular uptake assay was performed on two different cell lines, L132 and A549. Cellular uptake efficiencies of CS-TMZ-NP and CS-TMZ-FLA-NP were found to be concentration-dependent in both cell lines. CS-TMZ-FLA-NP produced a 2.5 fold greater accumulation of TMZ than CS-TMZ-NP in both cell lines. CS-TMZ-FLA-NP maintained a significantly higher deposition of TMZ in lung tissue (approximately 7.5 μg/g of lung tissue) when compared to free TMZ and CS-TMZ-NP. Mice treated with CS-TMZ-FLA-NP had a 100% survival rate with significant suppression of tumor growth. Histopathological and immunohistochemical studies also demonstrated that CS-TMZ-FLA-NP had superior anticancer activity compared to the other two treatments. Our results indicate that CS-TMZ-FLA-NP can effectively facilitate targeting to human lung cancer cell lines in vitro and to lung tumors in vivo in a sustained manner and so improve the therapeutic efficacy of TMZ.