MicroRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression. Hsa-miR-26b is an intronic miRNA located in the intron of CTDSP1 (carboxy-terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1). In the present study, the expression of hsa-miR-26b was examined during human preadipocyte differentiation. 15 days after induction of differentiation, mature human adipocytes were treated with adipokines. Hsa-miR-26b was differentially expressed during human preadipocyte differentiation. Tumor necrosis factor-α (TNF-α), leptin and resistin, but not interleukin-6, caused downregulation of hsa-miR-26b expression in adipocytes. These results suggest that the expression of hsa-miR-26b is affected by TNF-α, leptin and resistin and that hsa-miR-26b may be an important mediator in regulating the obesity-related insulin sensitivity and inflammatory responses.