AbstractC4photosynthesis evolved repeatedly from the ancestral C3state, improving photosynthetic efficiency by ∼50%. In most C4lineages photosynthesis is compartmented between mesophyll and bundle sheath cells but how gene expression is restricted to these cell types is poorly understood. Using the C3modelArabidopsis thalianawe identifiedcis-elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressedMYB76gene we identified a region necessary and sufficient for expression containing twocis-elements associated with the MYC and MYB families of transcription factors.MYB76expression is reduced in mutant alleles for each. Moreover, down-regulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with evolution of C4photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.