Aliphatic glucosinolates are a large group of plant secondary metabolites characteristic of Brassica, including model plant Arabidopsis. The diverse and complex degradation products of the aliphatic glucosinolates contribute to the plants' responses to herbivory, pathogen attack and environmental stresses. Most of the biosynthesis genes in aliphatic glucosinolate pathway had been cloned, and the research focus recently shifted to the regulatory mechanisms controlling aliphatic glucosinolates accumulation. Until now, more than 40 transcriptional regulators were identified to regulate the aliphatic glucosinolates pathway, however much more novel regulators likely remain to be discovered based on the research evidences over the past decade. In this current study, we took a systemic approach to functionally test 155 candidate transcription factors identified by yeast one-hybrid assay, and successfully validated at least 30 novel regulators that could significantly influence the accumulation of aliphatic glucosinolates in our experiment setup. We also showed that the regulators of aliphatic glucosinolates pathway have balanced positive and negative effects, and the glucosinolates metabolism and plant development could coordinate together. Our work is the largest scale effort so far to validate transcriptional regulators of a plant secondary metabolism pathway, and provides new insights of how the highly diverse plant secondary metabolisms are regulated at transcriptional level.