Recent studies of chloroplast-localized Sec14-like protein (CPSFL1, also known as phosphatidylinositol transfer protein 7, PITP7) showed that CPSFL1 is necessary for photoautotropic growth and chloroplast vesicle formation in Arabidopsis (Arabidopsis thaliana). Here, we investigated the functional roles of CPSFL1/PITP7 using two A. thaliana mutants carrying a putative null allele (pitp7-1) and a weak allele (pitp7-2), respectively. PITP7 transcripts were undetectable in pitp7-1 and less abundant in pitp7-2 than in the wild-type (WT). The severity of mutant phenotypes, such as plant developmental abnormalities, levels of plastoquinone-9 (PQ-9) and chlorophylls, photosynthetic protein complexes, and photosynthetic performance, were well related to PITP7 transcript levels. The pitp7-1 mutation was seedling lethal and was associated with significantly lower levels of PQ-9 and major photosynthetic proteins. pitp7-2 plants showed greater susceptibility to high-intensity light stress than the WT, attributable to defects in nonphotochemical quenching and photosynthetic electron transport. PITP7 is specifically bound to phosphatidylinositol phosphates (PIPs) in lipid-binding assays in vitro, and the point mutations R82, H125, E162, or K233 reduced the binding affinity of PITP7 to PIPs. Further, constitutive expression of PITP7 or PITP7 in pitp7-1 homozygous plants restored autotrophic growth in soil but without fully complementing the mutant phenotypes. Consistent with a previous study, our results demonstrate that PITP7 is essential for plant development, particularly the accumulation of PQ-9 and photosynthetic complexes. We propose a possible role for PITP7 in membrane trafficking of hydrophobic ligands such as PQ-9 and carotenoids through chloroplast vesicle formation or direct binding involving PIPs.