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Quantitative trait loci (QTL) mapping studies enable identification of loci that are part of regulatory networks controlling various phenotypes. Detailed investigations of genes within these loci are required to ultimately understand the function of individual genes and how they interact with other players in the network. In this study, we use transgenic plants in combination with natural variation to investigate the regulatory role of the AOP3 gene found in GS-AOP locus previously suggested to contribute to the regulation of glucosinolate defense compounds. Phenotypic analysis and QTL mapping in F2 populations with different AOP3 transgenes support that the enzymatic function and the AOP3 RNA both play a significant role in controlling glucosinolate accumulation. Furthermore, we find different loci interacting with either the enzymatic active or the RNA of AOP3 and thereby extend the regulatory network controlling glucosinolate accumulation.