Abstract: Objective To investigate the changes in mitochondrial calcium and extracellular sodium concentrations in the masseter muscle of rats with occlusal interference and the regulatory mechanism of mitochondrial Ca2+ overload by calmodulin kinase II (CaMK II). Methods SD rat models of occlusal interference were established by placing a stainless steel segments (0.8 mm in diameter) to raise the occlusal surface of the upper right first molar. At 3, 7, 14, and 21 days after occlusal interference and at 3 days after removal of occlusal interference, HE staining was used to observe the histomorphological changes of the masseter muscle. Mitochondrial calcium concentration in the masseter muscle was detected using fluorescence spectrophotometry, and direct turbidimetry with potassium pyroantimonate was used to detect the extracellular sodium concentration; the expression levels of masseter muscle p-CaMK II (Thr287) and CaMK II were detected using Western blotting. Results Compared with those in the corresponding control groups, mitochondrial Ca2 + concentration in the masseter muscle on occlusal interference side increased significantly at 3, 7, 14 and 21 days after occlusal interference (P<0.05), but was significantly lowered at 3 days after removal of the interference (P<0.05). The concentration of extracellular Na + increased progressively with time at 3, 7, 14 and 21 days after occlusal interference (P<0.05), and was significantly decreased at 3 days after interference removal (P<0.05). Occlusal interference for 3, 7 and 14 days resulted in significantly increased expressions of p-CaMK II (Thr287) and CaMK II (P<0.05), which was significantly decreased at 21 days compared with those in the control groups (P<0.05) and further decreased after removal of occlusal interference (P<0.05). Similar changes were also observed on the side without interference, but the changes on the interference side were more obvious (P<0.05). Conclusion Occlusal interference causes elevated mitochondrial Ca2 + and extracellular Na+ concentrations in the masseter muscle of rats to lead to calcium overload; the increase in mitochondrial Ca2 + concentration is correlated with the phosphorylation level of CaMK II signaling pathway, suggesting a negative feedback regulation mechanism by the CaMK II signal pathway.