Phosphorus (P) and iron (Fe) are two essential nutrients for plants, and their low availability in soil would seriously constrain crop growth and development, leading to compromised yield and quality. The down-regulation of genes involved in Fe uptake in phosphate (Pi)-deficiency were reported previously, but it is unclear whether there is a dependency on Fe concentration contributing to the disturbance. In this research, the variation of the transcript abundance of Fe acquisition genes upon Pi-deficiency with different concentrations of Fe was analyzed. Results showed that the pronounced inhibition of primary root growth under Pi-deficiency was significantly correlated to Fe concentrations, and the inhibition disappeared when Fe concentrations were decreased to a certain range. qRT-PCR analysis showed that the expression levels of Pi-deficiency marker genes IPS, SPX3, PHT1;4 were dramatically and exclusively induced by P-deficiency, which is independent of Fe concentrations. The expression levels of Fe-deficiency marker genes FRO2, IRT1 and CYP82C4 were significantly down-regulated by Pi-deficiency in a Fe-concentration-dependent manner, and decreased with increasing Fe concentrations, which might due to the elevated Fe availability in Pi-deficiency media. The results proposed a new perspective for management of P and Fe fertilizers in soil.