Inoculation of arbuscular mycorrhizal (AM) fungi has a great potential to reduce input of phosphorus fertilizer. In this study, we tested the hypothesis that transplanting of pre-inoculated plants (pre-inoculation) with AM fungal inoculum Glomus sp. strain R-10 (R-10) is more effective for increasing AM fungal colonization and soybean yield than placing R-10 inoculum into field soil (direct inoculation). We cultivated pre-inoculated and direct inoculated plants with and without R-10 in the same field. On the contrary to the hypothesis, hyphal colonization was increased by direct inoculation, but decreased by pre-inoculation in an early growth stage. Shoot phosphorus concentration, shoot dry weight, and yield also showed the same trend as the hyphal colonization. These results indicated that pre-inoculation with R-10 would be less effective for increasing AM fungal colonization and yield than direct inoculation. It may be due to a colonization strategy of R-10 and short duration for establishment of seedling.