Impairments in adiponectin multimerization lead to defects in adiponectin secretion and function and are associated with diabetes, yet the underlying mechanisms remain largely unknown. We have identified an adiponectin-interacting protein, previously named GST-kappa, by yeast 2-hybrid screening. The adiponectin-interacting protein contains 2 thioredoxin domains and has very little sequence similarity to other GST isoforms. However, this protein shares high sequence and secondary structure homology to bacterial disulfide-bond A oxidoreductase (DsbA) and is thus renamed DsbA-like protein (DsbA-L). DsbA-L is highly expressed in adipose tissue, and its expression level is negatively correlated with obesity in mice and humans. DsbA-L expression in 3T3-L1 adipocytes is stimulated by the insulin sensitizer rosiglitazone and inhibited by the inflammatory cytokine TNFalpha. Overexpression of DsbA-L promoted adiponectin multimerization while suppressing DsbA-L expression by RNAi markedly and selectively reduced adiponectin levels and secretion in 3T3-L1 adipocytes. Our results identify DsbA-L as a key regulator for adiponectin biosynthesis and uncover a potential new target for developing therapeutic drugs for the treatment of insulin resistance and its associated metabolic disorders.