Our study was conducted to investigate the effects of the fruits of Lycium chinense Mill. (Lycii Fructus, LF) and its bioactive compound, betaine, on muscle differentiation and mitochondrial biogenesis in C2C12 cells. LF extract and betaine was analyzed by high-performance liquid chromatography (HPLC). The expression of myosin heavy chain (MyHC) and peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α), sirtuin-1(Sirt-1), nuclear respiratory factor-1 (NRF-1), transcription factor A, mitochondrial (TFAM) and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), were determined in cellular or mitochondrial levels by quantitative polymerase chain reaction (qPCR) or Western blot, respectively. The glucose levels and total ATP contents were measured by the glucose consumption in a culture medium, cellular glucose uptake and ATP assays. LF extract at 4 mg/ml and betaine at 2 and 5 mM significantly increased the expression of MyHC in C2C12 myotubes, compared with non-treated cells. LF extract and betaine significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and TFAM mRNA and protein in the myotubes, as well as phosphorylation of AMPK and ACC. Furthermore, LF extract and betaine significantly increased the mitochondrial protein contents, as the TFAM and NRF-1 expressions were increased. LF extract and betaine also significantly increased the glucose uptake and ATP contents in the myotubes. The LF extract contained 3.18% betaine was quantitated by HPLC. LF extract and betaine enhanced muscle differentiation and energy metabolism through the up-regulation of mitochondrial biogenesis-regulating factors, suggesting that LF extract and betaine can help to prevent the dysfunction of skeletal muscle.