Intercropping is commonly practiced worldwide because of its benefits to plant productivity and resource-use efficiency. Belowground interactions in these species-diverse agro-ecosystems can greatly contribute to enhancing crop yields; however, our understanding remains quite limited of how plant roots might interact to influence crop biomass, photosynthetic rates, and the regulation of different proteins involved in CO₂ fixation and photosynthesis. We address this research gap by using a pot experiment that included three root-barrier treatments with full, partial and no root interactions between foxtail millet (Setaria italica (L.) P.Beauv.) and peanut (Arachis hypogaea L.) across two growing seasons. Biomass of millet and peanut plants in the treatment with full root interaction was 3.4 and 3.0 times higher, respectively, than in the treatment with no root interaction. Net photosynthetic rates also significantly increased by 112–127% and 275–306% in millet and peanut, respectively, with full root interaction compared with no root interaction. Root interactions (without barriers) contributed to the upregulation of key proteins in millet plants (i.e. ribulose 1,5-biphosphate carboxylase; chloroplast β-carbonic anhydrase; phosphoglucomutase, cytoplasmic 2; and phosphoenolpyruvate carboxylase) and in peanut plants (i.e. ribulose 1,5-biphosphate carboxylase; glyceraldehyde-3-phosphate dehydrogenase; and phosphoglycerate kinase). Our results provide experimental evidence of a molecular basis that interspecific facilitation driven by positive root interactions can contribute to enhancing plant productivity and photosynthesis.