WRKY transcription factors (TFs) have been proven to play vital roles in responses to biotic and abiotic stresses. The Lilium regale Wilson is a wild lily with a high level of resistance to fusarium wilt. A WRKY TF gene LrWRKY4 was cloned from L. regale Wilson by RTPCR based on previous transcriptome sequencing data in this study, and its function was analyzed to explore the transcriptional regulatory mechanism of L. regale during response to fusarium wilt pathogen Fusarium oxysporum infection. This study lays the foundation for subsequent further studying the function of the WRKY gene family in L. regale. The results showed that: (1) the open reading frame of LrWRKY4 was 993 bp, encoding 330 amino acids. LrWRKY4 contained a highly conserved ‘WRKYGQK’ heptapeptide and a C₂H₂ zinc finger motif, belonging to the group Ⅱc WRKY. (2) The GFPLrWRKY4 fusion vector was successfully constructed and transformed into onion by Agrobacterium tumefaciens, and the laser confocal microscopy observed that the green fluorescence expressed by the GFPLrWRKY4 fusion protein was specifically distributed in the nucleus of onion epidermal cells. (3) The overexpression vector pCAMBIA2300sLrWRKY4 was constructed and transformed into tobacco, and 11 lines of T₂ LrWRKY4 transgenic tobacco seedlings were obtained. There was no significant phenotype difference between transgenic tobacco and wildtype (WT) tobacco; the root and leaf inoculation assays showed that LrWRKY4 transgenic tobacco was more resistant to F. oxysporum than the WT; qRTPCR analysis showed that the LrWRKY4 gene of L. regale was expressed in 11 transgenic tobacco lines, and the expression of JA/SA signaling pathwayrelated genes were upregulated in LrWRKY4 transgenic tobacco compared with WT as well as some pathogenesisprotein related genes and antioxidantrelated genes. (4) The decay and lesion area of L. regale scales infected with LrWRKY4 RNAi vector were much larger than those of RNAi empty vector infected scales; the expression level of LrWRKY4 in L. regale scales transiently expressing LrWRKY4 RNAi vector was decreased by 45.7% compared with the control, and that was decreased by 93.8% after inoculation with F. oxysporum for 72 h. The expression levels of some JA/SA signaling pathwayrelated genes were significantly decreased in L. regale scales after transient expression of LrWRKY4 RNAi fragment. The results showed that L. regale LrWRKY4 encodes a class Ⅱc WRKY transcription factor, which is localized in plant cell nucleus; the LrWRKY4 was stably expressed in transgenic tobacco, and overexpression of LrWRKY4 can improve the resistance of tobacco to F. oxysporum. However, transient expression of LrWRKY4 RNAi fragment decreased the expression of JA/SA signaling pathwayrelated genes and enhanced the sensitivity of L. regale to F. oxysporum. It is speculated that LrWRKY4 gene is a positive regulator of the defense response against F. oxysporum in L. regale, and LrWRKY4 may induce the expression of defenserelated genes to regulate the resistance to F. oxysporum by participating in JA/SA mediated signal transduction pathways.