Objective Although catharanthine (1) is well known as a biosynthetic precursor of the anticancer alkaloid, vinblastine, its alternative metabolic pathways are unclear. Results Biotransformation of 1 by suspension-cultured cells of Catharanthus roseus gave a new oxidative-cleavage product (2). The structure of 2 was determined as 3-hydroxy-4-imino-catharanthine by spectroscopic methods. Maximum conversion (9.75 %) of 2 was observed after 120 h adding 6 mg of 1/100 ml to 12-day-old suspension-cultured cells of C. roseus. Furthermore, qRT-PCR experiment was performed to reveal the effect of 1 on the expression of the genes in the biosynthetic pathway of TIA 1 up-regulated the transcript level of D4H whilst down-regulating the transcript levels of G10H, LAMT, GES, and IRS. Conclusion A new metabolite of catharanthine, 3-hydroxy-4-imino-catharanthine, is reported.