The potency of cancer immunotherapy can be enhanced by administration of high-avidity ligands specific to receptors expressed on T cells. Antibodies or cytokines are the main agents used in such capacity. Antibody-mediated inhibition of cytotoxic T cell antigen-4 (CTLA-4) function in mice augments antitumor immunity and could serve as an important adjunct in cancer immunotherapy. However, antibody-based therapy used in the setting of chronic diseases such as cancer poses significant cost, manufacturing, and regulatory challenges. Here we describe the development of RNA aptamers that bind CTLA-4 with high affinity and specificity. These aptamers inhibit CTLA-4 function in vitro and enhance tumor immunity in mice. Moreover, assembly of the aptamers into tetrameric forms significantly enhances their bioactivity in vitro and in vivo. These results demonstrate that aptamers can be used to manipulate the immune system for therapeutic applications and that multivalent versions of aptamers may be particularly potent agents in vivo.