In Myxococcus xanthus, trehalose is synthesized in response to osmotic stress and during spore formation. Genome data analysis indicated that M. xanthus has five related enzymes involved in four trehalose synthesis pathways (OtsAB, TreYZ, TreT and TreS). Under osmotic conditions, the amount of trehalose in wild-type cells was increased quickly by the OtsAB pathway, and the otsAB mutant began to synthesize trehalose using the TreYZ pathway. Also, in comparison with the wild-type strain, the otsAB and treZ mutants showed similar to 65% and 40% reductions in the levels of intracellular trehalose accumulation, respectively, after 42 h of treatment with NaCl. In starvation-induced development of the wild-type strain, OtsAB activity was detected at middle and late stages of fruiting body development, and TreYZ activity was also detected at a late stage of development. Accumulation levels of trehalose in otsAB and treZ mutants were similar to 30% and 65% of that of the wild-type strain after 7 days of development, respectively. Wild-type and mutant strains did not show TreT and TreS activities under osmotic stress and development conditions. These results suggested that the OtsAB pathway may play a major role in trehalose biosynthesis in M. xanthus cells, with the TreYZ pathway playing an auxiliary role.