Flowering is a crucial stage for plant reproductive success; therefore, the regulation of plant flowering has been widely researched. Although multiple well-defined endogenous and exogenous flowering regulators have been reported, new ones are constantly being discovered. Here, we confirm that a novel plant growth regulator guvermectin (GV) induces early flowering inArabidopsis. Interestingly, our genetic experiments newly demonstrated thatWRKY41and its homologWRKY53were involved in GV-accelerated flowering as positive flowering regulators. Overexpression ofWRKY41orWRKY53resulted in an early flowering phenotype compared to the wild type (WT). In contrast, thew41/w53double mutants showed a delay in GV-accelerated flowering. Gene expression analysis showed that flowering regulatory genesSOC1andLFYwere upregulated in GV-treated WT,35S:WRKY41, and35S:WRKY53plants, but both declined inw41/w53mutants with or without GV treatment. Meanwhile, biochemical assays confirmed thatSOC1andLFYwere both direct targets of WRKY41 and WRKY53. Furthermore, the early flowering phenotype of35S:WRKY41lines was abolished in thesoc1orlfybackground. Together, our results suggest that GV plays a function in promoting flowering, which was co-mediated by WRKY41 and WRKY53 acting as new flowering regulators by directly activating the transcription ofSOC1andLFYinArabidopsis.