Lignin and its related metabolites play critical roles in plant growth and development. Thus, lignin biosynthesis has attracted interest as a novel target site of plant growth inhibitors. Chalcone has been shown to not only inhibit lignin biosynthesis in plants, but also to suppress the growth of many annual plant species. In order to know the direct effect of chalcone on plant metabolism, the effects of chalcone on the activities of key enzymes in lignin biosynthesis and on the related metabolites were clarified with a time-course study by using light-induced suspension cultures of soybean cells. The fresh weight and packed cell volume of the soybean cells were inhibited after 8 h of chalcone treatment. The activities of phenylalanine ammonia lyase (EC 22.214.171.124) and 4-coumarate: coenzyme A ligase (4CL; EC 126.96.36.199) were largely inhibited 4 h after the treatment with 0.15 mmol L−1 chalcone. Unlike these two enzymes, the activity of cinnamyl alcohol dehydrogenase (EC 188.8.131.52) was not inhibited until 16 h after the chalcone treatment. The content of the 4CL substrates and lignin in the soybean cells became relatively lower than the control under the light condition within 4 h and 8 h after the chalcone treatment, respectively. These results suggest that the growth suppression of soybean cells is positively associated with the inhibition of lignin biosynthesis by exogenous chalcone.