Auxin and cytokinin control callus formation from developed plant organs as well as shoot regeneration from callus. Dedifferentiation and regeneration of plant cells by auxin and cytokinin stimulation are considered to be caused by the reprogramming of callus cells, but this hypothesis is still argued to this day. Although an elucidation of the regulatory mechanisms of callus formation and shoot regeneration has helped advance plant biotechnology research, many plant species are intractable to transformation because of difficulties with callus formation. In this study, we identified fipexide (FPX) as a useful regulatory compound through a chemical biology-based screening. FPX was shown to act as a chemical inducer in callus formation, shoot regeneration and Agrobacterium infection. With regards to morphology, the cellular organization of FPX-induced calli differed from those produced under auxin/cytokinin conditions. Microarray analysis revealed that the expression of approximately 971 genes was up-regulated 2-fold after a 2 d FPX treatment compared with non-treated plants. Among these 971 genes, 598 genes were also induced by auxin/cytokinin, whereas 373 genes were specifically expressed upon FPX treatment only. FPX can promote callus formations in rice, poplar, soybean, tomato and cucumber, and thus can be considered a useful tool for revealing the mechanisms of plant development and for use in plant transformation technologies.