Abstract: MYB transcription factors play pivotal roles in plant metabolism and seed development. This study conducted a comprehensive investigation into the regulatory functions of these transcription factors in lipid synthesis and accumulation in Euphorbia lathyris L.. The analysis included genome-wide identification of MYB transcription factors, characterization of gene structures, physicochemical properties of encoded proteins, distribution of promoter cis-acting elements, and spatiotemporal expression patterns. The ElMYB114 coding sequence was cloned and genetically transformed into tobacco (Nicotiana tabacum L.) to characterize its biological function. Genome-wide identification revealed 199 MYB genes in E. lathyris, designated ElMYB1 to ElMYB199, all encoding proteins with SANT domains. The protein encoded by ElMYB114 was localized to the nucleus. Quantitative fluorescence qRT-PCR analysis showed that ElMYB114 expression peaked during the late stage of seed development. Overexpression of ElMYB114 in transgenic tobacco lines led to a 3.7% and 4.2% increase in total oil content in leaves and an 11.0% and 10.7% increase in total lipid content in seeds compared to the empty vector control. These findings suggest that ElMYB114 enhances carbon flux within the heterologous host toward lipid synthesis pathways, thereby promoting lipid accumulation in plant tissues. This study provides foundational insights into the MYB-mediated regulatory networks governing oil biosynthesis in E. lathyris.