Grain chalkiness is a major concern in rice production as it impacts grain yield and quality, eventually reducing its market value. High nighttime temperature (HNT) is a major inducer of grain chalk and most rice cultivars succumb to chalkiness under HNT, making it a major threat to rice production during climate change. A gene encoding the vacuolar H+ translocating pyrophosphatase (V-PPase) is a major quantitative trait locus of grain chalkiness in indica rice. Higher transcriptional activity of this gene is associated with increased chalk content. Although, natural variation in grain chalkiness of japonica rice has not been linked with V-PPase, the underlying mechanism is likely conserved between rice sub species. We carried out promoter targeting by CRISPR/Cas9 in japonica rice that correlated with downregulation of V-PPase in caryopses and reduced chalkiness in the mature grains. Most importantly, the targeted lines developed a significantly lower chalk under HNT. Biochemical analyses showed that suppression of V-PPase correlated with higher inorganic pyrophosphate and starch contents in the grains, and omics analysis showed that pathways related to starch and sugar metabolism were affected by V-PPase targeting. Our gene expression and biochemical data suggested that V-PPase targeting led to altered metabolic rate that supports more compact packing of starch granules under HNT conditions. Thus, HNT tolerance could be introduced by breeding suppression alleles of V-PPase.