Whether or not the atrophic skeletal muscle induces insulin resistance and its mechanisms are not resolved now. The antigravity soleus muscle showed a progressive atrophy in 1-week, 2-week, and 4-week tail-suspended rats. Hyperinsulinemic-euglycemic clamp showed that the steady-state glucose infusion rate was lower in 4-week tail-suspended rats than that in the control rats. The glucose uptake rates under insulin- or contraction-stimulation were significantly decreased in 4-week unloaded soleus muscle. The key protein expressions of IRS-1, PI3K, and Akt on the insulin-dependent pathway and of AMPK, ERK, and p38 on the insulin-independent pathway were unchanged in unloaded soleus muscle. The unchanged phosphorylation of Akt and p38 suggested that the activity of two signal pathways was not altered in unloaded soleus muscle. The AS160 and GLUT4 expression on the common downstream pathway also was not changed in unloaded soleus muscle. But the GLUT4 translocation to sarcolemma was inhibited during insulin stimulation in unloaded soleus muscle. The above results suggest that hindlimb unloading in tail-suspended rat induces atrophy in antigravity soleus muscle. The impaired GLUT4 translocation to sarcolemma under insulin stimulation may mediate insulin resistance in unloaded soleus muscle and further affect the insulin sensitivity of whole body in tail-suspended rats.