Background: New semi-synthetic aminoglycoside antibiotics generally use chemical modifications to avoid inactivity from pathogens. One of the most used modifications is 3’,4’-di-deoxygenation, which imitates the structure of gentamicin. However, the mechanism of di-deoxygenation has not been clearly elucidated. Results: Here, we report that the bifunctional enzyme, GenB4, catalyzes the last step of gentamicin 3’,4’-di-deoxygenation via reduction and transamination activities. Following disruption of gen B4 in wild-type M. echinospora , its products accumulated in 6’-deamino-6’-oxoverdamicin ( 1 ), verdamicin C2a ( 2 ), and its epimer, verdamicin C2 ( 3 ). Following disruption of gen B4 in M. echinospora △ gen K, its products accumulated in sisomicin ( 4 ) and 6’-N-methylsisomicin ( 5 , G-52). Following in vitro catalytic reactions, GenB4 transformed sisomicin ( 4 ) to gentamicin C1a ( 9 ) and transformed verdamicin C2a ( 2 ) and its epimer, verdamicin C2 ( 3 ), to gentamicin C2a ( 11 ) and gentamicin C2 ( 12 ), respectively. Conclusion: This finding indicated that in addition to its transamination activity, GenB4 exhibits specific 4’,5’ double-bond reducing activity and is responsible for the last step of gentamicin 3’,4’-di-deoxygenation. Taken together, we propose three new intermediates that may refine and supplement the specific biosynthetic pathway of gentamicin C components and lay the foundation for the complete elucidation of di-deoxygenation mechanisms.