Abstract: Rice allelopathy is a natural inhibitory phenomenon of some rice varieties against surrounding weeds. It is an eco-friendly method to control weeds, and it has become a popular research topic in the development of sustainable agriculture. Allelopathy is a quantitative trait regulated by a number of genes that are responsive to environmental changes. Previous studies have indicated that increasing the gene expression of the R2R3-MYB transcription factor MYB57 increases the expression of genes involved in the synthesis of phenolic acid allelochemicals, thereby enhancing the allelopathic effect of rice. This demonstrated that the transcription factor MYB57 efficiently regulates allelopathic weed suppression in rice; however, the regulatory network for MYB57 remains unknown. To further explore the transcriptional factors that regulate the gene expression of OsMYB57, an allelopathic rice accession (‘PI312777’) and a non-allelopathic accession (‘Lemont’) were used. The amplification of the promoter region of the OsMYB57 gene in ‘PI312777’ and ‘Lemont’ was conducted using specific biotin-labeled primers at the 5′ end. The proteins binding to the OsMYB57 gene promoter were co-precipitated using the DNA pull-down technique. Identification of these proteins using mass spectrometry showed that the gene expression of OsMYB57 was regulated by transcription factors (basic helix-loop-helix protein 009 bHLH009, LOC_Os04g32590.1, and LOC_Os02g31160.1) and transcription regulators (LOC_Os03g25430.1 and LOC_Os03g50110.1), which were encoded by the OsMYC2, Os04g0397500, Os02g0516800, Os03g0370250, and Os03g0708800 genes, respectively. The transcript level of these genes was up-regulated in ‘PI312777’ and ‘Lemont’ when they were co-cultured with barnyardgrass (Echinochloa crusgalli L.) for 1, 3, 5, and 7 days; more specifically, the expression of OsMYC2, Os02g0516800, and Os03g0708800 was significantly up-regulated in rice that was co-cultured with barnyardgrass for 5 days when compared to control groups. Among these proteins, basic helix-loop-helix protein 009 is a positive regulator of the jasmonate (JA) signaling pathway. The gene expression level of OsMYC2, Os04g0397500, Os02g0516800, Os03g0370250, and Os03g0708800 in ‘PI312777’ and ‘Lemont’ was up-regulated after the rice was treated with 0.05 mmol∙L⁻¹ of exogenous methyl jasmonate (MeJA), which resulted in an increased allelopathic suppression to barnyardgrass. Results of western blotting demonstrated that the protein expression levels of MYB57, MAPK11, and PAL2;3 in ‘PI312777’ and ‘Lemont’ were increased under the induction of MeJA. Furthermore, when compared with the control group of barnyardgrass treated independently with MeJA, the root exudates of ‘PI312777’ and ‘Lemont’ induced by 0.05, 0.10, and 0.20 mmol∙L⁻¹ of MeJA exhibited an enhanced growth inhibitory ratio, and the inhibitory effect of the root exudates of ‘PI312777’ induced by MeJA was higher than that of ‘Lemont’. The inhibitory ratios of ‘PI312777’ exudates induced by 0.05 mmol∙L⁻¹ of MeJA on the plant height and root length of barnyardgrass increased by 25.82% and 17.01%, respectively; while the same treatment using ‘Lemont’ root exudates showed a 5.30% and 15.36% increased inhibitory ratio on barnyardgrass. These results indicated that the gene expression of OsMYB57 was transcriptionally regulated by bHLH009 (MYC2), and that a suitable concentration of MeJA promoted the gene expression of OsMYC2, thereby enhancing OsMYB57 expression and resulting in an increase in MYB57, MAPK11, and PAL2;3 expression, which led to systematically up-regulated and improved rice allelopathic weed suppression.