Seeds of many brassicaceous plants accumulate high amounts of the soluble phenylpropanoid sinapine (sinapoylcholine). During early stages of seed germination sinapine is rapidly hydrolyzed by a sinapine esterase activity. From young seedlings of oilseed rape (Brassica napus) the sinapine esterase BnSCE3 was purified and characterized. Homology-based cloning led to the isolation of a full-length cDNA encoding BnSCE3. Sequence comparison identified BnSCE3 as a GDSL lipase like enzyme. In vitro assays of the BnSCE3 catalytic activity revealed a broad range of choline esters as potential substrates. Among these compounds, BnSCE3 showed highest catalytic efficiency with sinapine. Together with the expression profile, this indicates that BnSCE3 acts as a sinapine esterase in planta. In seeds of B. napus sinapine is the predominant phenolic compound in a complex group of antinutritive sinapate esters, which hamper the use of seed protein for food and feed. A strategy was developed to lower the sinapine content in B. napus by expressing the BnSCE3 gene under control of a seed-specific promoter. In transgenic BnSCE3 overexpressing seeds the sinapine content was less than 6% of the wild-type level. Metabolic profiling of transgenic seeds unraveled global effects on seed primary and secondary metabolism besides suppression of sinapine accumulation. The major agronomic traits like oil content, protein content and glucosinolate concentration were not altered.