The intricate assembly of photosystem I (PSI), a large multiprotein complex in the thylakoid membrane, depends on auxiliary protein factors. One of the essential assembly factors for PSI is encoded by ycf3 (hypothetical chloroplast reading frame number 3) in the chloroplast genome of algae and higher plants. To identify novel factors involved in PSI assembly, we constructed an epitope-tagged version of ycf3 from tobacco (Nicotiana tabacum) and introduced it into the tobacco chloroplast genome by genetic transformation. Immunoaffinity purification of Ycf3 complexes from the transplastomic plants identified a novel nucleus-encoded thylakoid protein, Y3IP1 (for Ycf3-interacting protein 1), that specifically interacts with the Ycf3 protein. Subsequent reverse genetics analysis of Y3IP1 function in tobacco and Arabidopsis thaliana revealed that knockdown of Y3IP1 leads to a specific deficiency in PSI but does not result in loss of Ycf3. Our data indicate that Y3IP1 represents a novel factor for PSI biogenesis that cooperates with the plastid genome-encoded Ycf3 in the assembly of stable PSI units in the thylakoid membrane.