MYB12 promotes flavonol biosynthesis in plants by targeting several early biosynthesis genes (EBGs) of this pathway. The transcriptions of these EBGs are also induced by sucrose signal. However, whether MYB12 is activated by sucrose signal and the other roles of MYB12 in regulating plant metabolism are poorly understood. In this study, two NtMYB12 genes were cloned from Nicotiana tabacum. Both NtMYB12a and NtMYB12b are involved in regulating flavonoids biosynthesis in tobacco. NtMYB12a is further shown to inhibit the accumulation of fatty acid (FA) in tobacco leaves and seeds. Posttranslational activation and chromatin immunoprecipitation assays demonstrate that NtMYB12a directly promotes the transcriptions of NtLOX6, NtLOX5, NtSFAR4, and NtGDSL2, which encode lipoxygenase (LOX) or lipase enzymes catalyzing the degradation of FA. NtLOX6 and NtLOX5 are shown to prevent the accumulation of FA in the mature seeds, and significantly reduced the percentage of polyunsaturated fatty acids (PUFAs) in tobacco. Sucrose stimulates the transcription of NtMYB12a, and loss function of NtMYB12a partially suppresses the decrease of FA content in tobacco seedlings caused by sucrose treatment. The regulation of sucrose on the expression of NtLOX6 and NtGDSL2 genes is mediated by NtMYB12a, but those of NtLOX5 and NtSFAR4 genes are independent of sucrose.