Central metabolism is a coordinated network that is regulated at multiple levels by resource availability and by environmental and developmental cues. Its genetic architecture has been investigated by mapping metabolite quantitative trait loci (QTL). A more direct approach is to identify enzyme activity QTL, which distinguishes between cis-QTL in structural genes encoding enzymes and regulatory trans-QTL. Using genome-wide association studies, we mapped QTL for 24 enzyme activities, nine metabolites, three structural components, and biomass in Arabidopsis thaliana. We detected strong cis-QTL for five enzyme activities. A cis-QTL for UDP-glucose pyrophosphorylase activity in the UGP1 promoter is maintained through balancing selection. Variation in acid invertase activity reflects multiple evolutionary events in the promoter and coding region of VAC-INV. cis-QTL were also detected for ADP-glucose pyrophosphorylase, fumarase, and phosphoglucose isomerase activity. We detected many trans-QTL, including transcription factors, E3 ligases, protein targeting components, and protein kinases, and validated some by knockout analysis. trans-QTL are more frequent but tend to have smaller individual effects than cis-QTL. We detected many colocalized QTL, including a multitrait QTL on chromosome 4 that affects six enzyme activities, three metabolites, protein, and biomass. These traits are coordinately modified by different ACCELERATED CELL DEATH6 alleles, revealing a trade-off between metabolism and defense against biotic stress.