Gibberellin (GA) is considered an important growth regulator involved in many aspects of plant development. However, little is known about the relationship between GA and lignification. In this study, we analyzed the role of GA in tracheary element (TE) differentiation and lignification using a Zinnia elegans xylogenic culture. When gibberellic acid-3 (GA3) was exogenously supplied, a slight increase in the frequency of TE differentiation and a remarkable increase in lignin content were observed. Computer image analysis of individual TEs showed that the lignification level of each TE was significantly increased in the culture treated with GA3 compared with those of the control. In contrast, suppression of TE differentiation and lignification was observed when GA biosynthesis was inhibited by ancymidol, paclobutrazol, or uniconazole. This suppression was restored by the addition of GA3. These results suggest that GA plays an important role in TE differentiation, and even more so in lignification. When conditioned medium obtained after 120 h of control culture was analyzed by high-performance liquid chromatography, many lignin precursors were detected. However, these lignin precursors were greatly reduced in the GA-treated culture. This result suggests that GA promotes lignification by activating the polymerization of lignin precursors.