In land-plant chloroplasts, the grana play multiple roles in photosynthesis, including the potential increase of photosynthetic capacity in light and enhancement of photochemical efficiency in shade. However, the molecular mechanisms of grana formation remain elusive. Here, we report a novel gene, Grana-Deficient Chloroplast1 (GDC1), required for chloroplast grana formation in Arabidopsis (Arabidopsis thaliana). In the chloroplast of knockout mutant gdc1-3, only stromal thylakoids were observed, and they could not stack together to form appressed grana. The mutant exhibited seedling lethality with pale green cotyledons and true leaves. Further blue native-polyacrylamide gel electrophoresis analysis indicated that the trimeric forms of Light-Harvesting Complex II (LHCII) were scarcely detected in gdc1-3, confirming previous reports that the LHCII trimer is essential for grana formation. The Lhcb1 protein, the major component of the LHCIIb trimer, was substantially reduced, and another LHCIIb trimer component, Lhcb2, was slightly reduced in the gdc1-3 mutant, although their transcription levels were not altered in the mutant. This suggests that defective LHCII trimer formation in gdc1-3 is due to low amounts of Lhcb1 and Lhcb2. GDC1 encodes a chloroplast protein with an ankyrin domain within the carboxyl terminus. It was highly expressed in Arabidopsis green tissues, and its expression was induced by photosignaling pathways. Immunoblot analysis of the GDC1-green fluorescent protein (GFP) fusion protein in 35S::GDC1-GFP transgenic plants with GFP antibody indicates that GDC1 is associated with an approximately 440-kD thylakoid protein complex instead of the LHCII trimer. This shows that GDC1 may play an indirect role in LHCII trimerization during grana formation.