Therapeutic transgene expression from oncolytic viruses represents one approach to increasing the effectiveness of these agents as cancer therapeutics. In the case of the oncolytic adenovirus (Ad), however, the genomic packaging capacity is constrained. To address this, we explored whether a transposon-based system could identify sites in the viral genome where endogenous Ad promoters could drive transgene expression via splicing and still maintain the replication capacity of the virus. Using GFP as a reporter gene and an E3-deleted Ad genome as a target, we tested three splicing signals. RACE analysis confirmed that gene expression from the GFP-expressing Ads occurs via splicing and traced expression to the Ad major late promoter (MLP). Replacement of the GFP transposon by an equivalent splice acceptor-luciferase expression cassette in the same orientation confirmed that substitute transgenes are also expressed via splicing from the MLP. Interestingly, insertion of the substitute transgene in the opposite orientation also resulted in expression that, in some cases, originated from within the ITR region of the viral genome. In summary, splice acceptor sequences can be used to control transgene expression from endogenous Ad promoters and this represents a genomically economical approach to arming oncolytic Ads.