Abstract: Endosperm development plays a major role in determining rice grain quality and yield, whereas the mechanisms underlying the early developmental stages remain largely unknown. Grain size is also an important trait directly influencing rice yield and appearance quality. Here, we identified the b336 mutant that is defective in early grain development, characterized by elongated grains with floury and shrunken endosperm. Cytological analyses indicated defective cellularization and cell proliferation in early endosperm development of b336. The b336 mutation also decreased cell length but increased cell number/height of glumes and caryopses. Molecular cloning revealed that the b336 mutant contains a missense substitution in the β-tubulin protein OsTUB1. This dominant-negative mutation destabilized both OsTUB1 and α-tubulins post-transcriptionally and caused abnormal microtubule (MT) arrays in b336. Combined cellular, biochemical, and genetic evidence indicates that OsTUB1 acts synergistically with an MT-associated protein End-binding 1c (OsEB1c) to modulate endosperm development. Notably, OsEB1c overexpression rescued the floury phenotype of b336 endosperm and markedly reduced the abundance of mutant OsTUB1, suggesting that OsEB1c likely plays a quality control role in the assembly of mitotic MT arrays. OsTUB1 also interacted with OsEB1a to coordinately regulate grain size. Furthermore, partial functional redundancy was found between OsEB1a and OsEB1c. Strikingly, OsTUB1 or OsEB1c overexpression significantly enlarged grain size and enhanced grain quality. Natural variations in the promoter of OsTUB1 likely contribute to grain length variations across rice cultivars by modulating its expression. Together, our results identify OsTUB1-OsEB1c and OsTUB1-OsEB1a as distinct molecular modules that separately regulate endosperm development and grain size, and provide target genes for use in biotechnology to improve rice yield and quality.