The role of tobacco transcription factor WRKY4 in leaf development and biotic stress tolerance was studied using RNAi suppressed transgenic plants. The leaves were more numerous and wider in NtWRKY4 RNAi suppressed transgenic lines compared to the vector control, while the levels of miRNA166 and miRNA396 were reduced in suppressed lines. NtWRKY4 expression was markedly induced in response to salicylic acid (SA), but not to abiotic stresses. When infected by tobacco mosaic virus (TMV), the leaves of the transgenic plants were more twisted and displayed a more obvious mosaic pattern compared to those of vector-transgenic plants. Less TMV viral RNA accumulated in vector-transformed plants than in transgenic plants. The results indicate that NtWRKY4 is involved in leaf morphogenesis and antiviral defense, which is seldom seen in WRKY family members.