Aims To investigate the major micro-organisms, which were isolated from internal organs, related to tetrodotoxin (TTX)-accumulation of puffer fish Lagocephalus lunaris. Methods and Results Puffer fish Lagocephalus lunaris around Chang Island in the Gulf of Thailand were collected to examine TTX-accumulation and microbial load in internal organs. The nine predominant micro-organisms isolated from the internal organs were determined TTX and studied in relation to the TTX-accumulation of the puffer fish. Shewanella putrefaciens, a predominant bacterium and related to the TTX-accumulation of the puffer fish, was examined growth and TTX-production after culture in modified Zobell medium. The results revealed that the average TTX-accumulation of the puffer fish directly varied to the bacterium load periodically, year-round. Furthermore, it coincided with the growth and the TTX-production of the bacterium, which grew slowly but produced high TTX at low temperature. Conclusions Shewanella putrefaciens was a major bacterium relating to TTX-accumulation of puffer fish L.similar to lunaris. It resulted in high TTX-accumulation of the puffer fish at low temperatures of seawater. Significance and Impact of the Study Temperature affected growth and TTX-production of S.similar to putrefaciens that resulted in TTX accumulated in puffer fish L.similar to lunaris.