GK coding sequence of Pseudomonas syringae pv. phaseolicola strain NPS3121 was cloned by PCR with the primers designed according to reported 1448A glycerol kinase (GK) amino acid sequence in GenBank. Sequencing analysis showed that the length of the GK gene was 1 506 bp, which could encode a polypeptide containing 501 amino acids. Bio–informatics analysis indicated that GK of strain NPS3121 contained 469 amino acids, with molecular weight 55 800, enriched in alpha helix, and isoelectric point was 5.44. GK deletion mutants of strain NPS3121 were constructed by integrated mutation and the results showed that the growth rate of GK deletion mutants reduced 18% and 16%, respectively in M9 and KBM medium, compared with wild strain. In addition, the content of glycerol in GK deletion mutants increased 6 times, compared to the wild strain.