<P><B>Abstract</B></P><P>A novel strategy to regulate cofactor balance in vivo for whole‐cell biotransformation using a synthetic flavin analogue is reported. High efficiency, easy operation, and good applicability were observed for this system. Confocal laser scanning microscopy was employed to verify that the synthetic flavin analogue can directly permeate into <I>Escherichia coli</I> cells without modifying the cell membrane. This work provides a promising intracellular redox regulatory approach to construct more efficient cell factories.</P>