Lung cancer is one of the leading causes of death worldwide and the most common of all cancer types. Histone acetyltransferase 1 (HAT1) has attracted increasing interest as a potential therapeutic target due to its involvement in multiple pathologies, including cancer. Aptamers are single-stranded RNA or DNA molecules whose three-dimensional structure allows them to bind to a target molecule with high specificity and affinity, thus making them exceptional candidates for use as diagnostic or therapeutic tools. In this work, aptamers against HAT1 were obtained, subsequently characterized, and optimized, showing high affinity and specificity for HAT1 and the ability to inhibit acetyltransferase activity in vitro. Of those tested, the apHAT610 aptamer reduced cell viability, induced apoptosis and cell cycle arrest, and inhibited colony formation in lung cancer cell lines. All these results indicate that the apHAT610 aptamer is a potential drug for the treatment of lung cancer.