A factorial analysis was conducted to investigate the effects of different levels of photosynthetic photon flux (PPF) and CO2 concentration on the interactions between the vesicular–arbuscular endomycorrhizal fungus Glomus intraradices and potato plantlets (Solanum tuberosum) cultured in an in vitro tripartite system. We observed that CO2 enrichment from 350 to 10000 ppm stimulated root colonization by the fungus, and that this stimulation was more pronounced under high PPF (300 μmol m−2 s−1) than low PPF (60 μmol m−2 s−1). Consistent with these observations, the effects of G. intraradices on dry matter production in potato plantlets were strongly dependent on the CO2 and PPF levels during cultivation. There was no significant effect of the mycorrhizal fungus on dry matter production at 350 ppm of CO2. However, under the high CO2 concentration, mycorrhiza had opposite effects on dry matter production depending on the PPF: a decrease (−21%) and a stimulation (+25%) of dry matter production after 2 wk of growth under low and high PPF, respectively, were observed in presence of G. intraradices relative to plantlets grown in its absence. Furthermore, in mycorrhizal plantlets grown under high levels of both PPF and CO2, the chlorophyll and carotenoid contents as well as the quantum yields of photosynthetic electron transport and the photochemical quenching qP of the chlorophyll‐a fluorescence measured near the PPF during growth were all higher than in non‐infected plantlets. Our results therefore indicate that mycorrhizal G. intraradices can alleviate the down regulation of photosynthesis related to sink limitation, and its effect on dry matter production is strongly dependent on the levels of CO2 and PPF during growth which determine the balance between the photosynthetic carbon uptake by the plantlets and the carbon cost by the fungus.