C(4) photosynthesis presents a sophisticated integration of two complementary cell types, mesophyll and bundle sheath cells. It relies on the differential expression of the genes encoding the component enzymes and transporters of this pathway. The entry enzyme of C(4) photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is found exclusively in mesophyll cells, and the expression of the corresponding gene is regulated at the transcriptional level. In the C(4) dicot Flaveria trinervia, the mesophyll-specific expression of the C(4) PEPC gene (ppcA) depends on a 41-bp segment in the distal promoter region referred to as MEM1 (for mesophyll expression module1). Here, we show that a MEM1 sequence found in the orthologous ppcA gene from the C(3) species Flaveria pringlei is not able to direct mesophyll-specific gene expression. The two orthologous MEM1 sequences of F. pringlei and F. trinervia differ at two positions, a G-to-A exchange and the insertion of the tetranucleotide CACT. Changes at these two positions in the C(3) MEM1 sequence were necessary and sufficient to create a mesophyll-specificity element during C(4) evolution. The MEM1 of F. trinervia enhances mesophyll expression and concomitantly represses expression in bundle sheath cells and vascular bundles.