Antisense oligonucleotides (ODN) technology is one of the most promising therapeutic strategies to prevent the progress of diseases through inhibiting the specific gene expression. They are well established to serve as molecular tools for several biologic applications, from the study of single gene function up to complex target validations. From the theoretical simple action, sequence-specific inhibition of mRNA functions after complex formation and presumably enzymatic degradation of the target mRNA, they obviously carry a high therapeutic potential to treat human diseases. In addition to the potential for the treatment, antisense ODN may be applicable for investigations of the mechanism and stereochemistry of biochemical reactions, mapping of nucleic acid protein interactions, and diagnostic applications. However, the design of antisense ODN, is very difficult because many factors affecting their activity and stability must be considered. Especially, the modifications of ODN are very critical and many researchers are trying to establish ODN which have resistance to nucleolytic degradation, high affinity to complementary nucleic acid, high selectivity in binding with complementary nucleic acid, the ability to activate ribonuclease H that selectively degrades the RNA strand of ODN-RNA complex, cell permeability, and favorable pharmacokinetic and pharmacodynamic attributes. In this review we would like to introduce some modifications of ODN design and examples of our applications of antisense ODN in cardiovascular disease in animal models.