In a key step in pre-mRNA splicing, the spliceosome recognizes the intronic 3' splice site (3’SS). Alternative usage of 3’SSs yields different mRNA isoforms that are essential for various cellular processes. How 3’SS selection is regulated to control gene expression is currently under investigation. In this study, I investigated the role of RNA secondary structure and the constitutive splicing factor Prp22, an RNA-dependent DExH ATPase, in 3’ splice site choice. Previously, I provided evidence that Prp22 rejects suboptimal 3’ splice sites by pulling on the splicing substrate. Here, I hypothesized that this pulling activity would also disrupt RNA secondary structure and thereby impact 3’SS selection, because RNA secondary structure can either repress a 3’SS by sequestering the site or activate a distal 3’SS by juxtaposing the 3’SS with the catalytic core of the spliceosome. To test our hypothesis in budding yeast, I assayed the impact of Prp22 on 3’SS selection in the context of RNA secondary structure in two native substrates. Indeed, Prp22p activated a 3’ splice site within a stem loop and repressed a 3’ splice site downstream of a stem loop. Mutational perturbation of the stems established that Prp22 destabilized the stems. Importantly, I observed that Prp22, through an impact on secondary structure, modulated alternative 3’ splice site choice. Our findings indicate that wildtype Prp22 unwinds RNA secondary structure in the region of a 3’ splice site and thereby activates a sequestered 3’SS and represses a distal 3’SS. This study identifies Prp22 as a potential target in the regulation of 3’ splice site selection.