In this work, we investigated the effects of pH and nitrogen forms on iron homeostasis and the expression profiles of genes involved in iron uptake and metabolism using tomato cultivar T3238 and its iron-inefficient mutant T3238fer. We showed that high external pH led to increased expression of four iron uptake genes (LeIRT1, LeIRT2, LeFRO1, LeNRAMP1) regardless of the nitrogen sources. Interestingly, the transcript level of FER was decreased at high pH and increased at low pH. In iron-inefficient mutant T3238fer, the expression of LeFRO1, LeIRT1 and LeNRAMP1 was much less than wild type under the culture conditions with high pH and on the non-buffered agar medium with NO(3) (-) as the sole N source, demonstrating that FER protein is required for the increased expression of LeFRO1, LeIRT1 and LeNRAMP1 under culture conditions with high pH. Considering the paradoxical expression patterns of FER to LeFRO1, LeIRT1 and LeNRAMP1 in T3238, we speculate that FER is essential, but is not the limited factor for the transcriptional regulation of the three iron uptake genes. In conclusion, the alteration of rhizosphere pH by assimilating NO(3) (-) or NH(4) (+) influenced Fe availability and consequently affected iron homeostasis in tomato. The enhanced expression of LeFRO1, LeIRT1 and LeNRAMP1 under the culture condition with high pH or on agar media with NO(3) (-) as the sole N source might be a consequence of reduced iron availability in the solution or agar medium at high pH.