Unloading skeletal muscle results in atrophy and weakness. Inhibition of calpain activity during unloading reduced atrophy, but the impact on force generation has not been determined. Our hypothesis was that inhibition of calpain, through muscle specific over-expression of calpastatin, would prevent the disruption of sarcomere structure and decreased specific force (kN/m²) observed during unloading. Calpastatin over-expressing (cp) and wild type (wt) mice were subjected to 3, 9, or 14 days of hindlimb suspension (HS). Compared with soleus muscles of non-suspended control mice, soleus muscles of wt mice showed a 25% decline in mass after 14 days of HS while maximum isometric force (Po) decreased by 40%, resulting in a specific P_o that was 35% lower than control values. Over the same time period, muscles of cp mice demonstrated 25% declines in both mass and P_o but no change in specific P_o. Consistent with the preservation of specific force during HS, soleus muscles of cp mice also maintained a high degree of order in sarcomere structure, in contrast to wt muscles that demonstrated misalignment of z-lines and decreased uniformity of thick filament lengths. Susceptibility to lengthening contraction-induced injury increased with the duration of HS and was not different for muscles of cp and wt mice. We conclude that inhibition of calpain activity during unloading preserves sarcomere structure such that the isometric force generating capability is not diminished, while the effects of unloading on lengthening contraction-induced injury likely occurs through calpain-independent mechanisms.