The non-specific lipid transfer proteins (nsLTPs) are able to bind various hydrophobic compounds and facilitate the transport of fatty acids between intracellular membranes, and nsLTPs are found in rice endosperm and embryo during seed development. However, whether nsLTPs function as lipid carriers and thereby affect lipid metabolism in rice grains remains unclear. To elucidate whether nsLTPs influence fatty acid distribution in rice, we generated OsLTP10-OE (OsLTP10 overexpression) and OsLTP10-CR (OsLTP10 CRISPR/Cas9) lines. Phenotypic and metabolic analyses indicated that OsLTP10 expression is closely associated with fatty acid (FA) profiles and grain appearance. In general, total fatty acid content in the brown rice of OsLTP10-OE was higher than that in wildtype, but OsLTP10-CR was lower than wildtype. While FA accumulation was altered in both tissues, the endosperm (milled grain) was more severely affected than the bran, with individual FAs in the milled grains of OsLTP10-OE expanding by 31.87–52.00%. Additionally, key grain quality traits were substantially altered; OsLTP10-CR lines displayed a significantly enlarged white-belly chalkiness area alongside a 19.50% reduction in amylose content, whereas OsLTP10-OE lines showed decreased chalkiness and a 7.80% increase in amylose. Overall, the fatty acid content and composition, chalkiness, brown rice size, and amylose were influenced by OsLTP10.