Iron is an essential element for plant growth and development, playing important roles in a variety of cellular activities including cell respiration, chlorophyll biosynthesis, and DNA synthesis. In chloroplasts, iron is essential for photosynthetic electron transport and functions as a cofactor for superoxide dismutases. Thus, iron homeostasis is critical for chloroplast and plant development. To better understand the mechanisms by which iron is transported into chloroplasts, we cloned and characterized the Nicotiana tabacum homolog of AtPIC1, which is a chloroplast iron transporter in Arabidopsis thaliana. NtPIC1 was expressed in many tissues of the tobacco plant, and the encoded protein was localized to the chloroplast envelope. Southern blotting indicated that there is a single copy of NtPIC1 in the tobacco genome. Moreover, yeast complementation assays suggested that NtPIC1 transports iron. We used RNA interference to downregulate NtPIC1 in transgenic plants, which resulted in albinism, dwarfism, iron-deficient chloroplasts, and chloroplast that exhibited ultrastructural defects. NtPIC1 overexpression resulted in deep-green leaves, elevated levels of chlorophyll, more densely packed chloroplasts, and the accumulation of iron and starch grains within chloroplasts. In addition, NtPIC1 influenced the expression of genes involved in iron transport, iron storage, iron oxidative stress, and the biogenesis of iron-sulfur proteins. These results suggest that NtPIC1 transports iron into chloroplasts, regulates iron homeostasis, and influences plant development.