Skeletal muscle atrophy is associated with many diseases including cancer, inflammatory diseases, neuromuscular diseases, and acute critical illness. Justicia procumbens L. has been used as a herbal remedy, but the pharmacological effect of J. procumbens on muscle atrophy has not yet been reported. Herein, we investigate the anti-atrophic effect of the n-butanol fraction of J. procumbens (JPBuFr) on dexamethasone (DEX)-induced muscle atrophy in C2C12 myotubes. The myotubes diameter, MHC positive area, ROS production, and mitochondria contents were observed under a fluorescence microscope, and various proteins related to degradation or synthesis were analyzed by western blots. JPBuFr significantly attenuated a reduction of myotube diameter, mitochondrial content, ATP level, myosin heavy chain, and myogenin expression induced by DEX. Furthermore, co-treatment of DEX and JPBuFr not only increased phosphorylation of Akt, mTOR, and p70S6K proteins but also decreased reactive oxygen species production and expression of protein degradation factors (MuRF1, Atrogin-1, FoxO3a) compared to DEX treatment. These results suggest that JPBuFr may provide potential protective effects against muscle atrophy, giving it potential for the development of anti-atrophic health functional foods.