This study investigated the mechanism of oleic acid (OA)-induced disassembly of myofibrils in cardiomyocytes. OA treatment disrupted myofibrils, as revealed by the disorganization of several sarcomeric proteins. Since focal adhesions (FAs) are implicated in myofibril assembly, we examined structural changes in FAs after OA treatment. Immunofluorescence studies with antibodies against FA proteins (vinculin, integrin β1D, and paxillin) showed that FAs and costameres disintegrated or disappeared after OA treatment and that the changes in FA proteins occurred prior to myofibril disassembly. The effects of OA on FAs and myofibrils were reversed after removal of OA. OA decreased expression of integrin β1D, paxillin, vinculin, and actin, and induced tyrosine dephosphorylation of FA kinase (FAK) and paxillin. These effects were blocked by pretreatment with sodium orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor. This inhibitor also prevented OA-induced myofibril disassembly, indicating the involvement of PTP in myofibril disassembly. Furthermore, OA increased protein levels of PTP-PEST. The upregulation of this phosphatase correlated with the tyrosine dephosphorylation of paxillin and FAK, which are targets for PTP-PEST. In addition, OA decreased RhoA activity and the phosphorylation of cofilin, a downstream target of RhoA. Cofilin dephosphorylation increased its actin-severing activity and led to the depolymerization of F-actin, which might provide another potential mechanism for OA-induced myofibril disassembly.