The UvrB subunit is a central component of the UvrABC incision complex and plays a pivotal role in damage recognition, strand excision and repair synthesis. A conserved structural motif (the SxSx motif) present in UvrB is analogous to a similar motif (TxGx) in the helicases of superfamily 2, whose function is not fully understood. To elucidate the significance of the SxSx (Ser143-Val144-Ser145-Cys146) motif in Mycobacterium tuberculosis UvrB (MtUvrB), different variants of MtUvrB subunit were constructed and characterized. The SxSx motif indeed was found to be essential for MtUvrB function: while Ser143 and Cys146 residues within this motif were crucial for MtUvrB function, Ser145 plays an important but less essential role. The SxSx motif-deleted mutant was drastically attenuated and three single (S143A, S145A and C146A) mutants and a double (S143A/S145A) mutant exhibited various degrees of severity in their DNA-binding, DNA helicase and ATPase activities. Taken together, these results highlight a hitherto unrecognized role for SxSx motif in the catalytic activities of UvrB.