Antisense oligonucleotides (ASOs) are versatile agents for controlling the translation and splicing of mRNA. ASOs have the potential to control the expression of every gene, providing a versatile platform for the development of therapeutic agents. Unlocking this potential requires understanding the mechanisms of action of ASOs. Most ASOs function by one of two mechanisms. Some recruit RNase H and promote cleavage of an RNA target. Others bind and block RNAs to prevent translation or redirect alternative splicing. Understanding the mechanism is crucial to optimization of function and the design of effective ASOs as experimental tools and therapeutics.