Plants exploit an array of defences against insect herbivores based on chemical and biomechanical properties. There is increasing evidence that plant toughness comprises a particularly effective defence against herbivory, yet studies to date have focussed exclusively on leaf toughness and folivore behaviour. The relationship between root mechanical properties and the chewing behaviour of a root-feeding insect, the Agriotes spp. wireworm (Coleoptera: Elateridae L.), feeding on tobacco (Nicoiana tabacum) is investigated. Root toughness is manipulated using introduced transgenes for the down-regulation of key enzymes in the lignin biosynthesis pathway: cinnamoyl-CoA reductase (CCR line) and caffeate O-methyltransferase and cinnamyl alcohol dehydrogenase (CO line). Two biomechanical analyses (i.e. conventional cutting and notched tensile) are conducted to quantify root toughness on both lines. Roots from the CO line are significantly tougher than those of the CCR line in terms of fracture toughness and fracture energy, although not for cutting energy or stiffness. Bioassays that compel wireworms to chew through roots demonstrate that only 30% can penetrate roots of the CO line compared with 90% on the CCR line. It takes wireworms over twice as long to penetrate roots from the CO line (8 h) compared with CCR roots (3.5 h). There is a statistically significant positive relationship between penetration time and fracture toughness evaluated with tensile tests, although not with cutting energy from cutting tests. Using this exploratory model system, it is concluded that root toughness derived from tensile tests is a practical indicator of the ability of root-feeding insects to penetrate roots.