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Simple Summary: Mitigating the possible adverse environmental impacts caused by transgene flow from genetically engineered crops to their wild/weedy relatives is an ideal strategy for resolving biosafety problems. To explore a transgene mitigation system in rice, we edited the seed-shattering genes (SH4 and qSH1) of a weedy rice line with strong seed shattering. The results showed substantially reduced seed shattering in the gene-edited weedy rice lines. The single gene-edited weedy rice lines displayed an inconsistent reduction in seed size-related traits. Reduced seed shattering was closely linked with a lack of abscission layers and reduced abscisic acid (ABA). In addition, most examined genes that were closely associated with ABA biosynthesis, ABA signaling transduction, and cell wall hydrolysis were downregulated in the gene-edited weedy rice lines. These findings can assist in exploring the underlying mechanisms of reduced seed shattering in weedy rice plants, in addition to practical applications for mitigating environmental impacts caused by transgene flow and for controlling the infestation of weedy rice. Mitigating the function of acquired transgenes in crop wild/weedy relatives can provide an ideal strategy to reduce the possible undesired environmental impacts of pollen-mediated transgene flow from genetically engineered (GE) crops. To explore a transgene mitigation system in rice, we edited the seed-shattering genes, SH4 and qSH1, using a weedy rice line ("C9") that originally had strong seed shattering. We also analyzed seed size-related traits, the total genomic transcriptomic data, and RT-qPCR expression of the SH4 or qSH1 gene-edited and SH4/qSH1 gene-edited weedy rice lines. Substantially reduced seed shattering was observed in all gene-edited weedy rice lines. The single gene-edited weedy rice lines, either the SH4 or qSH1 gene, did not show a consistent reduction in their seed size-related traits. In addition, reduced seed shattering was closely linked with the weakness and absence of abscission layers and reduced abscisic acid (ABA). Additionally, the genes closely associated with ABA biosynthesis and signaling transduction, as well as cell-wall hydrolysis, were downregulated in all gene-edited weedy rice lines. These findings facilitate our deep insights into the underlying mechanisms of reduced seed shattering in plants in the rice genus Oryza. In addition, such a mitigating technology also has practical applications for reducing the potential adverse environmental impacts caused by transgene flow and for managing the infestation of weedy rice by acquiring the mitigator from GE rice cultivars through natural gene flow. [ABSTRACT FROM AUTHOR]