Adiponectin is a pleiotropic protein secreted from adipocytes and renowned for its salutary effects. It forms a highly active high molecular weight (HMW) species that is paradoxically decreased in obesity. Efficient secretion of HMW adiponectin is dependent on post-translational modification (PTM) by hydroxylation and glycosylation of conserved lysines in the adiponectin collagenous domain. This work aims to characterize the pathway responsible for the PTM of adiponectin, and to define its significance in relation to obesity. siRNA mediated knockdown of GLT25D1 promoted lipid accumulation and increased adiponectin secretion in adipocytes with no change in PPARγ mRNA. Investigations into the mechanism of adiponectin multimerisation were continued in Adipo-HEK cells. siRNA mediated knockdown of LH3 and GLT25D1 indicated that LH3 was required for the secretion of total and HMW adiponectin whereas GLT25D1 was only required for the secretion of HMW adiponectin. Transient expression demonstrated that the intracellular retention of LH3 was dependent on GLT25D1. GLT25D1 was detected as a full length 68kDa protein and cleavage products, 50kDa-60kDa in size, with products present in the SGBS adipocytes and in mouse adipose tissue. Only 68kDa GLT25D1 co-immunoprecipitated with adiponectin and this was confirmed using mass-spectrometry. Prolonged high fat feeding in mice caused a decrease in the 68kDa protein and a disproportionate increase in the smaller cleavage products. Increased degradation of the 68kDa GLT25D1 with obesity may inhibit the secretion of HMW adiponectin and promote lipogenesis in obesity.