The Synechococcus sp. PCC 7942 spermidine synthase encoded by spds gene ( Synpcc7942_0628) is responsible for spermidine biosynthesis. Two Synechococcus strains, the overexpressing spds ( OX-spds) and the spds knockout ( Delta spds), were constructed and characterized for their growth and photosynthetic efficiency under osmotic stress imposed by sorbitol. The growth of Dspds was completely inhibited when cells were grown in the presence of 400 mM sorbitol. Under the same condition, the OX-spds showed a slightly higher growth than the wild type. The OX-spds under osmotic stress also had a significant increase of spermidine level in conjunction with the up-regulation of the genes involved in spermidine biosynthesis. A higher ratio of spermidine to putrescine, an index for stress tolerance, under osmotic stress was found in the OX-spds strain than in the wild type. Overall results indicated that the spermidine synthase enzyme plays an essential role in the survival of Synechococcus sp. PCC 7942 under osmotic stress.