The shape and size of grape are critical external quality traits that directly determine their commercial value. Although NF-Y family members are known to regulate fruit shape, their specific roles in grapes remain poorly understood. Here, we identified 31
NF-Y family genes in grape. Systematic analysis of their phylogeny, gene structures, and conserved motifs highlighted the evolutionary conservation of this family. Phylogenetic comparison of the NF-YA subfamily across
Arabidopsis, rice, tomato, and grape revealed that
VvNF-YA3 and
VvNF-YA5 are orthologs of
SlNF-YA8. Expression profiling showed that
VvNF-YA3 expression peaks at anthesis and three days after anthesis. Heterologous overexpression of
VvNF-YA3 in K326 tobacco significantly reduced leaf length, leaf shape index, corolla tube length, and pod dimensions. Moreover, its overexpression in
Nicotiana benthamiana resulted in larger but fewer leaf cells, accompanied by the downregulation of cell cycle genes responsible for cell division. Mechanistically, we identified VvSKIP34 as a VvNF-YA3-interacting protein and
VvIQD8 as its downstream target gene. Overexpression of
VvIQD8 in K326 tobacco yielded narrower leaves and pods, while significantly increasing corolla tube length, pod length, and fruit shape index. Collectively, these findings identify a VvNF-YA3-VvSKIP34-VvIQD8 module that controls cell division and fruit shape in tobacco, providing a functional link between
VvNF-YA3 and
VvIQD8 and suggesting a potential regulatory mechanism for fruit morphogenesis in grape.
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