The soils derived from ultramafic bedrock give rise to unique associations of landscape and plant community. Distinctive plant community in serpentine areas is derived from the extreme soil conditions that are low in nutrition (e.g., Ca) and high in toxic metals (e.g., Ni). While serpentine soils have been studied extensively, little study has appeared on the direct speciation of nickel naturally present in serpentine soils using synchrotron-based XAFS. The objectives of this study were to investigate the nutrient status and chemical speciation of Ni in several serpentine soils in Japan. Our study found that serpentine soils contained excess Mg and were deficient in available forms of Ca, which was indicated by the Ca/Mg ratio with <1.0. According to a sequential extraction analysis, the predominant Ni fraction was residual (~50%) and oxide occluded forms (~25%) whereas exchangeable Ni was negligible with <1% of the total proportion. The EXAFS investigation on soil Ni speciation showed the presence of Ni/Al-LDH phases and as Ni sorbed with humus, suggesting that the significant amount of Ni in serpentine soils was associated with secondary minerals and organic matter. The XAFS results showed that Ni hydroxides, oxides and carbonates did not appear to be important phases in these serpentine soils. Based on XAFS investigations and soil chemical properties, Ni toxicity to plants occurs unlikely but Ca deficiency may be significant in these serpentine soils.