Ex-situ conservation, the transplanting of individuals outside of their natural range, can dramatically alter biotic interactions among species. Here, we explored the shifts in the herbivory pressure and defense strategies of ex-situ populations of five endangered tree species (Pterostyrax psilophyllus, Davidia involucrata, Dipteronia sinensis, Tapiscia sinensis, and Tetracentron sinense) after 20-35 years since translocation. All species were not native to the ex-situ site and only P. psilophyllus had a closely related native species (Pterostyrax corymbosus). We compared the leaf damage type, herbivory rate, and chemistry (total phenols, tannins, N, and P) of each species, and assessed the relationship between the herbivory rate and leaf chemistry between the ex-situ and wild populations. Excluding P. psilophyllus, all species in the ex-situ populations showed changes in the type of leaf damage and lower overall herbivory rates. Ex-situ populations of P. psilophyllus showed reduced total phenol, N, and P levels, while the other four species showed decreased total phenol and tannin levels, or increased N and P levels. The herbivory rate was positively correlated with the N and P levels in wild populations, and negatively correlated with the total phenol content in ex-situ populations. These differences reflect the reduced herbivory pressure, decreased defense ability, and shift in defense strategy of ex-situ populations. We showed that prolonged exposure to the ex-situ environment led to the maladaptation of these endangered plant species to herbivory pressure in the natural environment, and that these shifts should be considered in ex-situ conservation and reintroduction strategies. ?