With the aim of increasing the cysteine level in rice (Oryza sativa L.) and thus improving its nutritional quality, transgenic rice plants were generated expressing an Escherichia coli serine acetyltransferase isoform (EcSAT), the enzyme synthesizing O-acetylserine, the precursor of cysteine. The gene was fused to the transit peptide of the Arabidopsis Rubisco and driven by a ubiquitin promoter to target the enzyme to plastids. Twenty-two transgenic plants were examined for transgene protein expression, and five lines with a high expression level and enzymatic activity, respectively, were selected for further analysis. In these lines, the contents of cysteine and glutathione increased 2.4-fold and 2-fold, respectively. More important is the increase in free methionine and methionine incorporated into the water-soluble protein fraction in seeds. Free methionine increased in leaves up to 2.7-fold, in seeds up to 1.4-fold, and bound to seed proteins up to 4.8-fold, respectively, while the bound methionine level remained constant or even decreased in leaves. Notably, the transgenic lines exhibited higher isoleucine, leucine, and valine contents (each up to 2-fold depending on tissue, free, or bound), indicating a potential conversion of methionine via methionine -lyase to isoleucine. As the transgenic rice plants overexpressing EcSAT had significantly higher levels of both soluble and protein-bound methionine, isoleucine, cysteine, and glutathione in rice they may represent a model and target system for improving the nutritional quality of cereal crops.