Intermuscular fat is essential for enhancing the flavor and texture of cultured meat. Mesenchymal stem cells derived from intermuscular adipose tissues are a source of intermuscular fat. Therefore, as a step towards developing a platform to derive intermuscular fat from mesenchymal stem cells (MSCs) for insertion between myofibrils in cultured beef, an advanced protocol of intermuscular adipose tissue dissociation effective to the isolation of MSCs from intermuscular adipose tissues was developed in cattle. To accomplish this, physical steps were added to the enzymatic dissociation of intermuscular adipose tissues, and the MSCs were established from primary cells dissociated with physical step-free and step-added enzymatic dissociation protocols. The application of a physical step (intensive shaking up) at 5 minutes intervals during enzymatic dissociation resulted in the greatest number of primary cells derived from intermuscular adipose tissues, showed effective formation of colony forming units-fibroblasts (CFU-Fs) from the retrieved primary cells, and generated MSCs with no increase in doubling time. Thus, this protocol will contribute to the stable supply of good quality adipose-derived mesenchymal stem cells (ADMSCs) as a fat source for the production of marbled cultured beef.