AZI1 (AZELAIC ACID INDUCED 1) of Arabidopsis thaliana encodes a 16.76 kDa protein with multiple functions, including resistances to low temperature and fungal infection. In the present work, the influences of AZI1 on lignin biosynthesis were investigated with overexpressing, T-DNA knockout and RNA interference lines. Western blotting coupled with immunolocalization exhibited that AZI1 and its paralog EARLI1 were expressed mainly in vascular tissues of inflorescence stems and leaves, but not in roots. Biochemical analyses and microscopic observation showed that knockdown and knockout of AZI1 led to decrease of the lignin content, reduction of the thickness of secondary wall, deformation of xylem cells and increase of the degradability by cellulase. In contrast to this, overexpression of AZI1 resulted in thicker cell wall and enhanced deposition of lignin in the lignified tissues. In comparison to wild-type plants and AZI1 overexpressing lines, the secondary wall of interfascicular fibers and xylem cells in AZI1 mutants showed yellowish-brown coloration after staining with Maule reagent, indicating the deposition of syringyl unit in lignin was reduced and the ratio of guaiacyl/syringyl monolignol units was increased when AZI1 was disrupted. RT-PCR analyses revealed that the transcription of CCR1 and CCOAOMT1, two genes associated with lignin synthesis, were repressed in T-DNA knockout lines. It has been shown that AZI1 probably modulates production and/or translocation of a long-distance signal during systemic acquired resistance. Our results suggested that AZI1 might be involved in regulation of lignin biosynthesis by influencing the expression of cinnamoyl-CoA reductase and caffeoyl-CoA O-methyltransferase.