Hypercholesterolemia, which is closely related to gallbladder bile stasis, can cause sphincter of Oddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO). Intracellular calcium ion concentration ([Ca2+]i) could influence the tension of SO. The β1 subunit of the big-conductance Ca2+sensitive K+ channel (BKCa) can enhance the sensitivity of the BKCa channel to [Ca2+]i. Absence and decline of the BKCa channel subunit β1 could lead to many diseases. However, the relationship between hypercholesterolemia and the expression of β1 subunit is not well understood. In this study, we successfully expressed and purified the rabbit BKCa β1 subunit protein and prepared its polyclonal antibody. The specificity of the prepared antibody was determined by weste blotting. A SOD rabbit model induced by a high cholesterol diet was established and the expression of the β1 subunit of SO was determined by immunohistochemical staining and weste blotting. Compared with the controls, our results demonstrated that hypercholesterolemia could decrease the expression of the β1 subunit in the SO cells from rabbits. This indicates that lower expression of BKCa channel β1 subunit might induce SOD.