The Cauliflower mosaic virus 35S promoter (P35S) induces transgene expression with insufficient activity and stability in some plant species, including lettuce. To develop a system to provide sufficient gene expression, a polyubiquitin promoter (PLsUbi) and terminator (TLsUbi) were isolated from lettuce, and this system was functionally compared with the conventional P35S-NOS terminator (P35S-Tnos) system by using a β-glucuronidase (GUS) reporter gene. In transgenic Arabidopsis, PLsUbi induced higher GUS activity than P35S, and the PLsUbi-TLsUbi combination induced higher GUS activity compared with the PLsUbi-Tnos combination, suggesting that the polyubiquitin terminator promotes transgene expression in concert with PLsUbi. The PLsUbi-TLsUbi combination induced less accumulation of GUS mRNA but > 10-fold higher GUS enzyme activity than the P35S-Tnos combination, suggesting that the PLsUbi-TLsUbi combination translationally promoted GUS expression in Arabidopsis. In transgenic lettuce, PLsUbi-TLsUbi transcriptionally and translationally promoted GUS expression, inducing approximately 16-fold-higher accumulation of GUS mRNA and > 800-fold-higher GUS enzyme activity compared with those induced by P35S-Tnos. Bisulfite sequencing methylation analysis of the introduced promoter sequences indicated that, for PLsUbi, the mean percentage of methylated cytosines in lettuce was 3.5 times that in Arabidopsis. For P35S, the mean percentage of methylated cytosines in lettuce was > 10 times that in Arabidopsis, and this methylation may be a major reason underlying the transcriptional inactivation of P35S in lettuce. Together, our results indicate that PLsUbi-TLsUbi promotes transgene expression in lettuce and Arabidopsis and may have broad applications in genetic engineering of additional plant species.