To investigate the effects of a compound (FF16), compatibility of Rhodiola crenulata, Cordyceps militaris, and Rheum palmatum, on insulin resistance. The results showed that FF16 significantly improved the insulin sensitivity through decreasing AUC values in insulin tolerance tests by 24.1%, 38.5%; reducing the levels of serum insulin by 46.0%, 30.4%, of HOMA-IR by 52.4%, 81.2%; and reversing the lower GIR values by 119.3%, 202.4% in IRF mice and KKAy mice, respectively. In addition, in KKAy mice, the value of whole body insulin sensitivity index (ISWBI) was enhanced by 1.0 times, the abilities of the insulin-induced glucose uptake in liver, adipose and skeletal muscle were enhanced by 1.5, 2.8 and 2.2 times, respectively, in FF16-treated mice comparing with those in model mice. The recombinant human protein tyrosine phosphatase 1B (PTP1B) activity was inhibited by FF16 in vitro with the IC50 value of 0.225 mg x L(-1). The increased PTP1B expression in the liver was also reversed by 45.8% with the administration of FF16 in IRF mice. In conclusion, FF16 could improve insulin resistance by inhibiting the activity of PTP1B.