Vitamin B12 (B12) is an essential micronutrient required for several metabolic reactions. Animal and clinical studies show that B12-deficiency is associated with metabolic syndrome. Given the key metabolic role of adipose tissue, we investigated whether B12 deficiency may affect triglyceride synthesis and lipid metabolism leading to adipose tissue inflammation. The AbdSc pre-adipocyte cell line (Chub-S7) and human AbdSc primary pre-adipocytes were differentiated under different B12 concentrations (25 pM, 100 pM, 1nM, 500 nM). Human Om, Sc-AT and blood samples were collected from 106 pregnant women at delivery. Serum B12 and relevant metabolic risk factors were measured. Gene expression was performed by q-RT-PCR, de novo triglyceride synthesis was quantified by radioactive tracing, ß-oxidation and palmitate-induced oxygen consumption rate was determined using the seahorse-XF analyzer. Adipocytes cultured in low-B12 conditions showed significantly increased expression (P<0.01) of triglyceride biosynthesis genes (ELOVL6,SCD,GPAT,LPIN1 and DGAT2), a significantly decreased expression (P<0.01)of ß-oxidation genes (FAT/CD36, CPT1-ß, ACADL, ECHS1 and ACAA2) and an increased expression (P<0.01) of pro-inflammatory cytokines (IL-1, IL-6,IL-8,IL-18,TGF-β,TNF-α and MCP-1). These data were also confirmed in the AT of B12-deficient pregnant women. Additionally, real-time fatty acid flux synthesis and fatty-acid-oxidation induced by palmitate were significantly altered (P<0.05) in B12-deficient adipocytes. Our data highlights that B12-deficiency has profound effects on adipocyte dysfunction, opening new insights into the pathogenesis of maternal obesity and the relevance of micronutrient supplementation for pregnant mothers.