Objective To investigate the changes of adenosine triphosphatase activity, expression of calmodulin (CaM), and calmodulin-dependent protein kinase II (CaMK II) protein in CaMK II signaling pathways in the liver tissue of rats with spleen-qi deficiency. Methods 3-month-old SPF Wistar rats (male:female=1:1) were randomly divided into 4 groups: normal control group, and three spleen-qi deficient model groups (observed on days 7, 14 and 21), 12 rats in each group. The spleen-qi deficient rat models were generated by bitter-cold purgatives, exhaustion and fully or poorly feeding food in turn, and their general conditions and ATPase activity were evaluated. Confocal laser scanning microscopy was used to detect cellular [Ca²⁺]i concentrations and Western blotting was used to test CaM, CaMK II, p-CaMK II expression in liver tissue of the spleen-qi deficient rats. Results Compared with the normal group, general condition was poor, activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were significantly decreased (P<0.01, P<0.05), and expressions of CaM, CaMK II, and p-CaMK II in the liver tissue were decreased. A most obvious difference was observed in the spleen-qi deficient model day 21 group (P<0.01). Conclusions Our results demonstrate that decease of adenosine triphosphatase activities and [Ca²⁺]i concentration is involved in the spleen-qi deficiency, and expressions of key molecules of CaMK II signaling pathway, CaM, CaMK II and p-CaMK II proteins, are also decreased.