The aim of this study was to evaluate the role of bamboo shoot fiber (BSF) in regulating insulin sensitivity in mice. Two BSFs were obtained from D. hamiltonii and D. latiforus. C57BL/6 mice were fed a low-fat diet or a high-fat diet with 10% fiber as cellulose (HFC), or two BSFs (HFDH or HFDL) for 13 weeks. Compared with the HFC group, HFDH and HFDL groups exhibited considerably less body weight gain, lower levels of fasting glucose and insulin, and lower values of glucose areas under the curve during glucose and insulin tolerance tests. BSF groups also had lower glucoses stimulated insulin concentrations. In addition, more phosphorylation of Akt in insulin target tissues was associated with higher protein expression levels of PGC-1 alpha and phosphorylated AMPK and p38 in BSF fed mice. These results indicated that BSF improved insulin sensitivity in high-fat diet-fed mice through enhancing insulin signaling and activating PGC-1 alpha.