Dirigent super-family abounds throughout the plant kingdom, especially vascular plants. To elucidate the function of cotton (Gossypium hirsutum) DIR genes in lignification, two cDNAs (designated GhDIR1 and GhDIR2) encoding putative dirigent proteins were isolated from cotton cDNA libraries. Real-time quantitative reverse transcription-polymerase chain reaction analysis revealed that GhDIR1 transcript was preferentially accumulated in cotton hypocotyls, whereas GhDIR2 was predominantly expressed in cotton fibers. Overexpression of GhDIR1 gene resulted in an increase in lignin content in transgenic cotton plants, compared with that of wild type. Histochemical assay revealed that the transgenic plants displayed more widespread lignification than that of wild type in epidermis and vascular bundle. Furthermore, the transgenic cotton plants displayed more tolerance to the infection of Verticillium dahliae. Our data suggest that GhDIR1 may be involved in cotton lignification which can block the spread of fungal pathogen V. dahliae.