Background Human subcutaneous fat tissue consists of two layers: superficial adipose tissue (SAT) and deep adipose tissue (DAT). Some recent reports suggest that a disproportionate accumulation of DAT is related to obesity-associated metabolic complications. However, the differences in adipocyte function between SAT and DAT are unclear. Materials and Methods To clarify the differences in human adipocyte characteristics between SAT and DAT, human ceiling culture-derived proliferative adipocytes (ccdPAs) were primary cultured from SAT and DAT of three lean females. Differences in adipogenic differentiation potential and sensitivity to exogenous adipogenic factors were examined. Epigenetic modification of the CpG island DNA methylation levels of genes related to adipogenesis was measured. Results In histological analyses, the mean adipocyte size in SAT was significantly larger than that in DAT (8741 ± 416 vs. 7732 ± 213 μm2, P < 0.05). Primary cultured adipocytes from SAT showed significantly greater adipogenesis than did those of DAT. Sensitivity to partial adipogenic stimulation was significantly different between ccdPAs of SAT and DAT. PPAR- protein expression and leptin protein secretion from ccdPAs were significantly higher in SAT than DAT. DNA methylation levels of PPAR- were significantly lower in ccdPAs of SAT than DAT. Conclusion Adipocyte size was larger in SAT than DAT in vivo. This is consistent with the findings of an in vitro study that, compared with ccdPAs in DAT, ccdPAs in SAT have higher adipogenic potential and lower DNA methylation levels of PPAR-.