Arabidopsis plastidic HSP90C is an HSP90 family molecular chaperone that is required for chloroplast development and function. To understand the mechanism of action of HSP90C within the chloroplast, we conducted a yeast two-hybrid screening and revealed it interacts directly with the photosystem II extrinsic protein PsbO1, which performs a canonical function in the thylakoid lumen. To understand the biological significance of HSP90C-PsbO1 interaction, we investigated the role of HSP90C in modulating the stromal and thylakoid distribution of PsbO1GFP fusion protein. Fusion to GFP significantly delays the PsbO1 thylakoid transport and induces a variegation phenotype. Overexpression of HSP90C promotes the thylakoid distribution of PsbO1GFP and alleviates the leaf variegation. By tracking the chloroplast maturation during photomorphogenesis, we observed PsbO1GFP tends to form distinct fluorescent clusters within the stroma with delayed thylakoid membrane biogenesis, while HSP90C overexpression corrects these adverse effects. We also demonstrated that active HSP90C function is specifically required for stable accumulation of mature PsbO1GFP in thylakoid by using specific inhibitor geldanamycin. This study therefore not only identified novel HSP90C interactors, but also reports for the first time that PsbO1 enroute from the cytoplasm to thylakoid lumen is tightly regulated by the HSP90C chaperone complex in plastid stroma; whereas the proper HSP90C homeostasis is also critical for chloroplast maturation and function.