Purpose: MicroRNAs (miRNA), single-stranded non-protein coding gene products, regulate gene expression through post-transcriptional inhibition, and known to be involved in essential biological processes including obesity and insulin resistance. This study was conducted to elucidate the role of miR100 expression in human adiposity and insulin resistance. Methods: Pair samples were obtained from subcutaneous (SAT) and visceral adipose tissue (VAT) during elective operation in 71 men and 42 women. Mature miRNA levels were determined by qRT-PCR and normalized to levels of U6 small nuclear ribonucleoprotein. Subcutaneous (SFA) and visceral fat area (VFA) was determined by abdominal multidetector CT scanning. Adipocyte sizing was determined by the osmium-fixed method on a Coulter counter. Results: Study 1 (clinical study): In men and women, SFA was larger than VFA, and the mean size of adipocytes was greater in SAT than in VAT. Level of log(miR100) was significantly decreased in VAT in men (p=0.025), but comparable between SAT and VAT in women. Level of log(miR100) in VAT was positively correlated with HOMA-IR, and tended to be positively correlated with expression level of macrophage marker, CD68. Level of log(miR100) in SAT was positively correlated with expression level of IL1β, NLRP3 and TLR4 in men but not in women. Study 2 (in vitro study): When stimulated with lipopolysaccharide (LPS, 1-10ng/mL), expression of miR100 was not changed in mouse macrophage-like Raw264.7 cells, but enhanced in adipocyte-like 3T3-L1 cells. A member of the NOX family of NADPH oxidases NOX4, which has a possible MiR100 target at 3'-UTR, was up-regulated in 3T3-L1 cells by LPS, but not changed in Raw264.7 cells. NOX2, another type of NADPH oxidase, was not detected in 3T3L1 cells but was in Raw264.7 cells. Conclusion: Adipose tissue miR100 is derived mostly from adipocytes and may play a direct or indirect role in regulating expressions of adipocyte inflammatory cytokines, adipocyte-specific NADPH oxidase, and insulin sensitivity in human.