As the human population grows rapidly, food shortages will become an even greater problem; therefore, increasing crop yield has become a focus of rice breeding programs. The maize gene,ZmDUF1645, encoding a putative member of the DUF1645 protein family with an unknown function, was transformed into rice. Phenotypic analysis showed that enhancedZmDUF1645expression significantly altered various traits in transgenic rice plants, including increased grain length, width, weight, and number per panicle, resulting in a significant increase in yield, but a decrease in rice tolerance to drought stress. qRT-PCR results showed that the expression of the related genes regulating meristem activity, such asMPKA,CDKA, a novel crop grain filling gene (GIF1), andGS3, was significantly changed in theZmDUF1645-overexpression lines. Subcellular colocalization showed thatZmDUF1645was primarily localized on cell membrane systems. Based on these findings, we speculate thatZmDUF1645, like theOsSGLgene in the same protein family, may regulate grain size and affect yield through the cytokinin signaling pathway. This research provides further knowledge and understanding of the unknown functions of the DUF1645 protein family and may serve as a reference for biological breeding engineering to increase maize crop yield.