The basic leucine zipper (bZIP) proteins are ubiquitous in plants and play important roles in plant defense responses. In this study, based on an expressed sequence tag from a suppression subtractive hybridization cDNA library of Lilium regale Wilson during Fusarium oxysporum f. sp. lilii infection, a novel bZIP transcription factor gene LrbZIP1 was isolated from L. regale root using the rapid amplification of cDNA ends method. The predicted protein of LrbZIP1 with 142 amino acid residues contains a basic domain signature and a leucine zipper motif. The quantitative reverse transcription-PCR (qRT-PCR) analysis showed that the transcription level of LrbZIP1 was higher in roots of L. regale than in young stems and leaves. Moreover, the expression of LrbZIP1 was up-regulated in the incompatible interaction between L. regale and F. oxysporum f. sp. lilii as well as after treatments with stress-related signaling molecules. To verify the function of LrbZIP1, a constitutive expression vector of LrbZIP1 was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The results of Southern blotting and qRT-PCR analyses demonstrated that the LrbZIP1 was integrated into genome of the tobacco transformants and highly expressed. Under normal conditions, the T1 transgenic tobacco lines showed higher antioxidant enzyme activities and transcription levels of several resistance-related genes than the wild type. Moreover, the T1 transgenic tobacco plants showed strong resistance to F. oxysporum f. sp. lilii infection.