We recently demonstrated that non-plant cells, yeast WAT21, produce a steroidal plant hormone, castasterone. To understand how castasterone is generated in WAT21 cells, deuterium-labeled (26, $28-^2H_6$) 6-deoxoteasterone, 6-deoxotyphasterol and 6-deoxocastasterone were fed to WAT21 cells, and their metabolites were analyzed by gas chromatography-mass spectrometry. When [$^2H_6$]-6-deoxoteasterone was fed, [$^2H_6$]-6-deoxo-3-dehydroteasterone and [$^2H_6$]-6-deoxotyphasterol were identified as metabolites. When [$^2H_6$]-6-deoxotyphasterol was used, [$^2H_6$]-6-deoxoteasterone and [$^2H_6$]-6-deoxo-3-dehydroteasterone and [$^2H_6$]-6-deoxocastasterone were detected. When [$^2H_6$]-6-deoxocastasterone was added, [$^2H_6$]-castasterone was identified. Taken together, a biosynthetic sequence, 6-deoxoteasterone${leftrightarrow}$6-deoxo-3-dehydroteasterone${leftrightarrow}$6-deoxotyphasterol${ ightarrow}$6-deoxocastasterone${ ightarrow}$castasterone seems to function to produce brassinosteroids in WAT21 cells. Coupled with the presence of a biosynthetic sequence, teasterone${ ightarrow}$3-dehydroteasterone${leftrightarrow}$typhasterol ${ ightarrow}$castasterone, this suggests that brassinosteroids in WAT21 cells were biosynthesized via the same kind of pathways established in plants. This study provides a clue for possible mass-production of brassinosteroids in non-plant cells, yeast WAT21 for commercial use in agriculture.