Shewanella putrefaciens is a well-known specific spoilage organism (SSO) and cold-tolerant microorganism in refrigerated fresh marine fish. Cold-adapted mechanism includes increased fluidity of lipid membranes by the ability to finely adjust lipids composition. In the present study, the lipid profile of S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C was explored using ultra-high-pressure liquid chromatography/electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to discuss the effect of lipid composition on cold-adapted tolerance. Lipidomic analysis detected a total of 27 lipid classes and 606 lipid molecular species in S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C. S. putrefaciens cultivated at 30 °C (SP-30) had significantly higher content of glycerolipids, sphingolipids, saccharolipids, and fatty acids compared with that at 0 °C (SP-0); however, the lower content of phospholipids (13.97%) was also found in SP-30. PE (30:0), PE (15:0/15:0), PE (31:0), PA (33:1), PE (32:1), PE (33:1), PE (25:0), PC (22:0), PE (29:0), PE (34:1), dMePE (15:0/16:1), PE (31:1), dMePE (15:1/15:0), PG (34:2), and PC (11:0/11:0) were identified as the most abundant lipid molecular species in S. putrefaciens cultivated at 30, 20, 10, 4, and 0 °C. The increase of PG content contributes to the construction of membrane lipid bilayer and successfully maintains membrane integrity under cold stress. S. putrefaciens cultivated at low temperature significantly increased the total unsaturated liquid contents but decreased the content of saturated liquid contents.