Iron (Fe) deficiency is a common mineral stress in plants, including sorghum. Although the soil fungus Trichoderma harzianum has been shown to mitigate Fe deficiency in some circumstances, neither the range nor mechanism(s) of this process are well understood. In this study, high pH-induced Fe deficiency in sorghum cultivated in pots with natural field soil exhibited a significant decrease in biomass, photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, and Fe-uptake in both the root and shoot. However, the establishment of T. harzianum colonization in roots of Fe-deprived sorghum showed significant improvements in morpho-physiological traits, Fe levels, and redox status. Molecular detection of the fungal ThAOX1 (L-aminoacid oxidase) gene showed the highest colonization of T. harzianum in the root tips of Fe-deficient sorghum, a location thus targeted for further analysis. Expression studies by RNA-seq and qPCR in sorghum root tips revealed a significant upregulation of several genes associated with Fe uptake (SbTOM2), auxin synthesis (SbSAURX15), nicotianamine synthase 3 (SbNAS3), and a phytosiderophore transporter (SbYS1). Also induced was the siderophore synthesis gene (ThSIT1) in T. harzianum, a result supported by biochemical evidence for elevated siderophore and IAA (indole acetic acid) levels in roots. Given the high affinity of fungal siderophore to chelate insoluble Fe3+ ions, it is likely that elevated siderophore released by T. harzianum led to Fe(III)-siderophore complexes in the rhizosphere that were then transported into roots by the induced SbYS1 (yellow-stripe 1) transporter. In addition, the observed induction of several plant peroxidase genes and ABA (abscisic acid) under Fe deficiency after inoculation with T. harzianum may have helped induce tolerance to Fe-deficiency-induced oxidative stress and adaptive responses. This is the first mechanistic explanation for T. harzianum's role in helping alleviate Fe deficiency in sorghum and suggests that biofertilizers using T. harzianum will improve Fe availability to crops in high pH environments.