Despite their economic and ecological importance, defense responses of conifers to pests are little understood. In a 3-year experiment, we monitored systemic fungal (Diplodia pinea)- and insect (Neodiprion sertifer)-induced defense protein activities and total soluble proteins in needles and phloem of Austrian pine (Pinus nigra) across a soil fertility gradient. In both years, total soluble protein content of foliage and phloem declined with increasing fertility across induction treatments, while defensive protein activities generally increased with increasing fertility. In 2005, total soluble protein content in branch phloem was increased by fungal inoculation of the stem. Peroxidase activity was suppressed in needles by insect defoliation in 2006, while polyphenol oxidase activity was systemically induced in branch phloem by insect attack in 2005. Trypsin inhibitor activities in phloem did not respond to any induction or fertility treatment. Nutritive quality of Austrian pine tissue declined with increasing fertility, while several protein-based defenses simultaneously increased.