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The economic value of Humulus lupulus L. (hop) is recognized, but the primary metabolism of the hop strobilus has not been quantified in response to elevated CO2. The photosynthetic contribution of hop strobili to reproductive effort may be important for growth and crop yield. This component could be useful in hop breeding for enhanced performance in response to environmental signals. The objective of this study was to assess strobilus gas exchange, specifically the response to CO2 and light. Hop strobili were measured under controlled environment conditions to assess the organ’s contribution to carbon assimilation and lupulin gland filling during the maturation phase. Leaf defoliation and bract photosynthetic inhibition were deployed to investigate the glandular trichome lupulin carbon source. Strobilus-level physiological response parameters were extrapolated to estimate strobilus-specific carbon budgets under current and future atmospheric CO2 conditions. Under ambient atmospheric CO2, the strobilus carbon balance was 92% autonomous. Estimated strobilus carbon uptake increased by 21% from 415 to 600 µmol mol−1 CO2, 14% from 600 to 900 µmol mol−1, and another 8%, 4%, and 3% from 900 to 1200, 1500, and 1800 µmol mol−1, respectively. We show that photosynthetically active bracts are a major source of carbon assimilation and that leaf defoliation had no effect on lupulin production or strobilus photosynthesis, whereas individual bract photosynthesis was linked to lupulin production. In conclusion, hop strobili can self-generate enough carbon assimilation under elevated CO2 conditions to function autonomously, and strobilus bracts are the primary carbon source for lupulin biosynthesis.