Most plants are known as C3 plants because the first product of photosynthetic CO2 fixation is a three-carbon compound. C4 plants, which use an alternative pathway in which the first product is a four-carbon compound, have evolved independently many times and are found in at least 18 families. In addition to differences in their biochemistry, photosynthetic organs of C4 plants show alterations in their anatomy and ultrastructure. Little is known about whether the biochemical or anatomical characteristics of C4 photosynthesis evolved first. Here we report that tobacco, a typical C3 plant, shows characteristics of C4 photosynthesis in cells of stems and petioles that surround the xylem and phloem, and that these cells are supplied with carbon for photosynthesis from the vascular system and not from stomata. These photosynthetic cells possess high activities of enzymes characteristic of C4 photosynthesis, which allow the decarboxylation of four-carbon organic acids from the xylem and phloem, thus releasing CO2 for photosynthesis. These biochemical characteristics of C4 photosynthesis in cells around the vascular bundles of stems of C3 plants might explain why C4 photosynthesis has evolved independently many times.