Telbivudine (LdT), a widely prescribed anti-hepatitis B virus (HBV) drug for the treatment of chronic Hepatitis B (CHB), causes adverse reactions ranging from creatine kinase (CK) elevation to myopathy. The purpose of this study was to explore the mechanism(s) of LdT induced CK elevation. The effects of LdT on mitochondrial morphology and proteins (TK2 and β-actin), oxidative stress, intracellular Ca2+ levels, Ca2+-related signaling pathway (CaMKK2/AMPK), and Ca2+-related biomarkers such as superoxide dismutase (SOD) and malondialdehyde (MDA) were assessed in human skeletal muscle cells (HSKMCs). The results showed that LdT induced a dose-dependent increase in CK activity in HSKMCs, without affecting mitochondrial morphology, and TK2 and β-actin protein levels, following 72 h of treatment. In addition, LdT increased Ca2+ production, ROS generation, MDA and lipid peroxide (LPO) levels, and activated the CaMKK2/AMPK signaling pathway. Moreover, these effects were attenuated by the BAPIA-AM (the calcium chelator). We also confirmed the presence of relevant markers (MDA, LPO, and SOD) in serum from CHB patients after LdT treatment, and found that CK was positively correlated with MDA and LPO, and negatively associated with SOD. These findings indicate that LdT induces CK elevation and oxidative stress associated with imbalance of intracellular Ca2+ in HSKMCs, suggesting that Ca2+/CaMKK2 axis imbalance may underlie human LdT-induced CK elevation. The present findings provide a solid basis for assessing the mechanism of drug-induced CK elevation, which can help develop new tools for the prevention and treatment of diseases associated with drug-induced CK elevation.