Arctic Mesorhizobium sp. N33 isolated from nodules of Oxytropis arctobia in Canada's eastern Arctic has a growth temperature range from 0 °C to 30 °C and is a well-known cold-adapted rhizobia. The key molecular mechanisms underlying cold adaptation in Arctic rhizobia remains totally unknown. Since the concentration and contents of metabolites are closely related to stress adaptation, we applied GC-MS and NMR to identify and quantify fatty acids and water soluble compounds possibly related to low temperature acclimation in strain N33. Bacterial cells were grown at three different growing temperatures (4 °C, 10 °C and 21 °C). Cells from 21 °C were also cold-exposed to 4°C for different times (2, 4, 8, 60 and 240 minutes). We identified that poly-unsaturated linoleic acids 18:2 (9, 12) & 18:2 (6, 9) were more abundant in cells growing at 4 or 10 °C, than in cells cultivated at 21 °C. The mono-unsaturated phospho/neutral fatty acids myristoleic acid 14:1(11) were the most significantly overexpressed (45-fold) after 1 hour of exposure to 4 °C. As reported in the literature, these fatty acids play important roles in cold adaptability by supplying cell membrane fluidity, and by providing energy to cells. Analysis of water-soluble compounds revealed that isobutyrate, sarcosine, threonine and valine were more accumulated during exposure to 4 °C. These metabolites might play a role in conferring cold acclimation to strain N33 at 4 °C, probably by acting as cryoprotectants. Isobutyrate was highly upregulated (19.4-fold) during growth at 4 °C, thus suggesting that this compound is a precursor for the cold-regulated fatty acids modification to low temperature adaptation.