Defects in insulin-stimulated glucose uptake in skeletal muscle result from the dysfunction of insulin signaling including the phosphatidylinositol-3 kinase (PI3K) pathway and a novel β-arrestin-2-mediated signaling, which leads to insulin resistance (IR). Pollen Typhae, a Chinese herb, has been used for thousands of years in traditional Chinese medicine, and has the potential to inhibit the development of IR. We have previously reported that Pollen Typhae total flavone (PTF), the extract from Pollen Typhae, ameliorates high-glucose- and high-insulin-induced impairment of glucose uptake in 3T3-L1 adipocytes, but the mechanisms remain unclear. The objective of this study was to investigate the effects of PTF on glucose uptake, and to explore the underlying mechanisms in C2C12 myotubes. PTF improved insulin-stimulated glucose uptake in a dose- and time-dependent manner in C2C12 myotubes, and prevented palmitate-induced IR. Furthermore, PTF enhanced the basal gene expression of Src and Akt2, elevated the protein expression of β-arrestin-2, Src and Akt, increased the phosphorylation of insulin receptor-β at Tyr1150/1151 and Akt at Thr308/Ser473 in an insulin-dependent manner, but had no effects on the protein expression of PI3K-p85 or the activity of PI3K. Inhibition of Src but not PI3K restrained PTF-induced phosphorylation of Akt and glucose uptake. Our findings indicate that PTF improves insulin-induced glucose uptake via the β-arrestin-2-mediated signaling in C2C12 myotubes.