A transient expression system based on a deleted version of Cowpea mosaic virus (CPMV) RNA-2, termed CPMV-HT, in which the sequence to be expressed is positioned between a modified 5′ UTR and the 3′ UTR has been successfully used for the plant-based expression of a wide range of proteins, including heteromultimeric complexes. While previous work has demonstrated that alterations to the sequence of the 5′ UTR can dramatically influence expression levels, the role of the 3′ UTR in enhancing expression has not been determined. In this work, we have examined the effect of different mutations in the 3′UTR of CPMV RNA-2 on expression levels using the reporter protein GFP encoded by the expression vector, pEAQexpress-HT-GFP. The results showed that the presence of a 3′ UTR in the CPMV-HT system is important for achieving maximal expression levels. Removal of the entire 3′ UTR reduced expression to approximately 30% of that obtained in its presence. It was found that the Y-shaped secondary structure formed by nucleotides 125–165 of the 3′ UTR plays a key role in its function; mutations that disrupt this Y-shaped structure have an effect equivalent to the deletion of the entire 3′ UTR. Our results suggest that the Y-shaped secondary structure acts by enhancing mRNA accumulation rather than by having a direct effect on RNA translation. The work described in this paper shows that the 5′ and 3′ UTRs in CPMV-HT act orthogonally and that mutations introduced into them allow fine modulation of protein expression levels.