This short communication is to report the inhibitory effect of deuterium oxide (D₂O) on germination and culturability of bacterial spore. Germination was triggered by L-alanine under ambient condition and in situ monitored by Tb³⁺-dipicolinic acid assay, phase contrast microscopy and absorbance measurement. Germination kinetics were characterized by final germination level, germination half time, and germination speed. The culturability of germinated spores was evaluated by enumerating colony-forming units. Results revealed that D₂O retarded the whole germination process and reduced the culturability but didn’t affect the final germination level. We postulate that D₂O enhanced the stability of some spore-specific signaling proteins.