The principal mechanism underlying the reduced rate of protein synthesis in atrophied skeletal muscle is largely unknown. eEF2k impairs the ability of eEF2 to bind to the ribosome via T56 phosphorylation. Perturbations in the eEF2k/eEF2 pathway during various stages of disuse muscle atrophy have been investigated utilizing a rat hindlimb suspension (HS) model. Two distinct components of eEF2k/eEF2 pathway misregulation were demonstrated, observing a significant (P<0.01) increase in eEF2k mRNA expression as early as 1-day HS and in eEF2k protein level after 3-day HS. We set out to determine whether eEF2k activation is a Ca2+-dependent process with involvement of Cav1.1. The ratio of T56-phosphorylated:total eEF2 was robustly elevated after 3-day HS, which was completely reversed by BAPTA-AM and decreased by 1.7-fold (P<0.05) by nifedipine. Transfection of C2C12 with pCMV-eEF2k and administration with small molecules were used to modulate eEF2k and eEF2 activity. Importantly, pharmacological enhancement of eEF2 phosphorylation induced P-p70S6k (T389) upregulation and restoration of global protein synthesis in the HS rats. Taken together, the eEF2k/eEF2 pathway is upregulated during disuse muscle atrophy involving calcium-dependent activation of eEF2k partly via Cav1.1. The study provides evidence, in vitro and in vivo, of the eEF2k/eEF2 pathway impact on p70S6k activity as well as protein expression of key atrophy biomarkers, MAFbx/atrogin-1 and MuRF-1.