Many herbivorous insects sequester plant defense compounds from their host plants to protect themselves from natural enemies. In plants, these defense compounds are often stored as protoxins separated from their activating enzymes. A well-known example is the glucosinolate-myrosinase defense system in plants of the order Brassicales. When plant tissue is ingested by herbivores, glucosinolates are hydrolyzed by the enzyme myrosinase to form highly reactive isothiocyanates. We previously reported that flea beetles of the genus Phyllotreta selectively sequester high amounts of glucosinolates from their crucifer host plants, and convergently evolved their own myrosinase which enables them to utilize sequestered glucosinolates for their own purposes (Beran et al., 2014). The presence of intact glucosinolates in these beetles suggests that despite tissue damage, ingested glucosinolates are not activated by the plant myrosinase. Rapid and efficient glucosinolate uptake from the gut lumen into gut epithelial cells can prevent hydrolysis and thus might be crucial to overcome this activated plant defense. We use the horseradish flea beetle Phyllotreta armoraciae as a model to study the molecular basis of sequestration in insects. In short-term feeding experiments, we showed that ingested glucosinolates are rapidly sequestered in the foregut. To identify the transporters that mediate glucosinolate import from the foregut lumen into gut epithelial cells, we focused on the MFS transporter family, which is known to transport a wide range of substrates. A phylogenetic analysis of putative MFS transporter sequences identified in P. armoraciae and other beetles revealed several specifically expanded clades in P. armoraciae. Out of 21 candidate genes that were heterologously expressed in Sf9 cells, nine showed glucosinolate transport activity in vitro. Interestingly, most candidate genes were exclusively expressed in the malpighian tubules, and two genes were additionally expressed in the foregut. We currently elucidate the function of these transporters in glucosinolate sequestration in vivo using RNAi. To better understand the function of sequestered glucosinolates, we performed bioassays with P. armoraciae larvae and the generalist predatory ladybird Harmonia axyridis. Upon predator attack, P. armoraciae larvae emitted high amounts of isothiocyanates and ladybird larvae stopped feeding within a few seconds and were highly irritated. However, silencing myrosinase gene expression in P. armoraciae larvae led to increased mortality compared to control larvae in survival assays with ladybird larvae. Our results demonstrate how Phyllotreta use plant defense metabolites to defend themselves against predators.