Clinical trials involving in patients with osteoporosis have reported that activated vitamin D₃ (1α,25(OH)₂D₃, calcitriol) can prevent falling by acting on the skeletal muscles. However, pharmacological mechanisms of 1α,25(OH)₂D₃ with respect to skeletal muscle hypertrophy or atrophy are still poorly understood. Therefore, we examined changes in the expression of several related genes in human myotubes to test whether 1α,25(OH)₂D₃ influences hypertrophy and atrophy of skeletal muscle. Myotubes treated with 1α,25(OH)₂D₃ increased interleukin-6 (IL-6) expression and inhibited expression of tumor necrosis factor alpha (TNF-α), whereas the expression of insulin-like growth factor-1 (IGF-1) that is involved in muscle hypertrophy was not affected. However, 1α,25(OH)₂D₃ treatment significantly inhibited the expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1), ubiquitin ligases involved in muscle atrophy. The analysis of pathways using microarray data suggested that 1α,25(OH)₂D₃ upregulates AKT-1 by inhibiting the expression of protein phosphatase 2 (PP2A), a phosphatase acting on AKT-1, in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, thereby inhibiting the expression of ubiquitin ligases. Thus, this study showed that 1α,25(OH)₂D₃ might have an inhibitory effect on the expression of MAFbx and MuRF1 in skeletal muscle and a suppressive effect on muscle degradation in patients with osteoporosis.