Low birth weight (LBW) is associated with increased risk of developing type 2 diabetes (T2D). The appetite-regulating hormone leptin is released from mature adipocytes and its production may be decreased in immature preadipocytes from LBW individuals. We recruited 14 men born with LBW and 13 controls born with normal birth weight (NBW). Biopsies were obtained from subcutaneous abdominal fat depots and preadipocytes were isolated and cultured. Gene expression of leptin and selected differentiation markers were analyzed during preadipocyte differentiation and cell culture media was collected to analyze leptin secretion. DNA methylation of CpG sites in the leptin promoter was measured using pyrosequencing. We found that differentiating preadipocytes from LBW individuals showed reduced leptin gene expression and a corresponding reduced leptin release compared to NBW individuals. Mean DNA methylation of the proximal LEP promoter was increased in LBW compared to NBW individuals. The notion of impaired adipocyte maturation in LBW individuals was supported by a lower mRNA expression of the differentiation markers; fatty acid binding protein 4 (FABP4), peroxisome proliferator-activated receptor γ (PPARγ), and glucose transporter type 4 (GLUT4). Our findings are consistent with impaired preadipocyte maturation contributing to increased risk of developing T2D in LBW subjects.