The delay in autumnal senescence that has occurred in recent decades has been linked to rising temperatures. Here, we suggest that increasing atmospheric CO₂ may partly account for delayed autumnal senescence and for the first time, through transcriptome analysis, identify gene expression changes associated with this delay. Using a plantation of Populus x euramericana grown in elevated CO₂ (eCO₂) with free-air CO₂ enrichment (FACE) technology, we investigated the molecular and biochemical basis of this response. A Populus cDNA microarray was used to identify genes representing multiple biochemical pathways influenced by eCO₂ during senescence. Gene expression changes were confirmed through real-time quantitative PCR, and leaf biochemical assays. Pathways for secondary metabolism and glycolysis were significantly up-regulated by eCO₂ during senescence, in particular, those related to anthocyanin biosynthesis. Expressed sequence tags (ESTs) representing the two most significantly up-regulated transcripts in eCO₂, LDOX (leucoanthocyanidin dioxgenase) and DFR (dihydroflavonol reductase), gave (eCO₂/ambient CO₂ (aCO₂)) expression ratios of 39.6 and 19.3, respectively. We showed that in eCO₂ there was increased autumnal leaf sugar accumulation and up-regulation of genes determining anthocyanin biosynthesis which, we propose, prolongs leaf longevity during natural autumnal senescence.