Periodic outbreaks of spruce budworm (SBW) affect large areas of ecologically and economically important conifer forests in North America, causing tree mortality and reduced forest productivity. Host resistance against SBW has been linked to growth phenology and the chemical composition of foliage, but the underlying molecular mechanisms and population variation are largely unknown. Using a genomics approach, we discovered a β-glucosidase gene,Pgβglu-1, whose expression levels and function underpin natural resistance to SBW in mature white spruce (Picea glauca) trees. In phenotypically resistant trees,Pgβglu-1transcripts were up to 1000 times more abundant than in non-resistant trees and were highly enriched in foliage. The encoded PgβGLU-1 enzyme catalysed the cleavage of acetophenone sugar conjugates to release the aglycons piceol and pungenol. These aglycons were previously shown to be active against SBW. Levels ofPgβglu-1transcripts and biologically active acetophenone aglycons were substantially different between resistant and non-resistant trees over time, were positively correlated with each other and were highly variable in a natural white spruce population. These results suggest that expression ofPgβglu-1and accumulation of acetophenone aglycons is a constitutive defence mechanism in white spruce. The progeny of resistant trees had higherPgβglu-1gene expression than non-resistant progeny, indicating that the trait is heritable. With reported increases in the intensity of SBW outbreaks, influenced by climate, variation ofPgβglu-1transcript expression, PgβGLU-1 enzyme activity and acetophenone accumulation may serve as resistance markers to better predict impacts of SBW in both managed and wild spruce populations.