Circular RNAs (circRNAs) are a type of noncoding RNA that are formed by back-splicing from eukaryotic protein-coding genes. The most frequently reported and well-characterized function of circRNAs is their ability to act as molecular decoys, most often as miRNA and protein sponges. However, the functions of most circRNAs still need to be better understood. To more fully understand the biological relevance of validated circRNAs, knockdown functional analyses can be performed using antisense oligonucleotides, RNA interference (RNAi) experiments (e.g., targeting back-splicing junction sites), the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)-9 system (e.g., generating circRNA-specific knockouts), and CRISPR-Cas13 technology to effectively target circRNAs without affecting host genes. In this review, I summarize the feasibility and effectiveness of circRNA knockdown through antisense strategies for investigating the biological roles of circRNAs in cultured cells and animal models.