To determine the impact of genotypic variation in secondary metabolite production on antiherbivore resistance and plant fitness, we genetically silenced biosynthetic genes for nicotine, trypsin proteinase inhibitors (TPI), and jasmonate (JA) production in two accessions of Nicotiana attenuata: one from Utah (UT) which responds to herbivory with JA-induced nicotine and TPI production, and one from Arizona (AZ) which is TPI-deficient but also produces JA-induced nicotine. Transient silencing of JA biosynthesis increased Manduca sexta larval growth on wild type (WT) plants of both accessions, but not on TPI-deficient UT or nicotine-deficient AZ lines, demonstrating that JA-mediated resistance to M. sexta requires TPIs in the UT and nicotine in the naturally TPI-deficient AZ accession. When transplanted into a native UT population, AZ and UT plants, rendered equally able or unable to produce nicotine and TPIs by stable transformation, received significantly different levels of herbivory. Both accessions differed in their resistance depending on the type of herbivores: resistance to rare, voracious herbivores (Saltatoria and Mammalia) was greater in AZ than UT lines, and dependent on nicotine production, while resistance to small, abundant herbivores (Coleoptera and Thysanoptera) was greater in UT lines, and dependent on TPI production. AZ lines produced more flowers and seed capsules than UT lines independently of TPI production costs. This fitness advantage was lost when accessions did not produce nicotine. We conclude that these two accessions have developed different survival strategies and thus differ in the cost-benefit functions of their JA-mediated defences.