There has been an increasing interest in soybean cultivation in hydroponic systems. Because soybean plants can utilize nitrogen derived from biological N2 fixation, the use of Rhizobium inoculated plants may eliminate or decrease the need for mineral nitrogen fertilization in hydroponic systems. Thus, the objective of this study was to establish effective methods for making inoculated soybean transplants for a substrate-based hydroponic system. Inoculated plants were effectively produced by rooting seedling stem cuttings in a medium containing a Rhizobium inoculant. We also investigated the effects of different nitrogen forms and sub-irrigated nutrient solution concentrations on the growth and root nodule formation of the inoculated plants. The application of a nitrogen-free nutrient solution had minimal positive effects on the growth and nodulation of the inoculated plants. Ammonium-fed plants lacked root nodules regardless of the ammonium concentration. Furthermore, a 50% nutrient solution combining with nitrate or urea as the nitrogen source did not promote root nodulation. Therefore, inoculated plants should be subirrigated with an approximately 25% nutrient solution supplemented with nitrate or urea to induce early growth and nodulation prior to transplantation.