Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutant. Additionally, the g-pifi mutant exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.