Serpentine grasslands support a unique endemic flora composed of many rare species that are tolerant of the inhospitable soil conditions characteristic of ultramafic soils. In the eastern United States, rare plants of many serpentine grasslands are being displaced as a result of invasion by Pinus species and consequent changes in environmental conditions. Community structure and function of symbiotic soil arbuscular mycorrhizal fungi (AMF) also may be affected by Pinus succession. This study examined changes in soil properties, AMF communities, and plant growth in serpentine grassland soils that have been invaded by Virginia pine (Pinus virginiana Mill.). Sites representing three vegetation types (grassland, pine savanna, and pine woodland) were assessed at the Soldiers Delight Natural Environmental Area, Maryland, USA. In comparison to grassland sites, soil pH and concentrations of Ca, Mg, and Ni were reduced in pine woodland soils. Savanna soils exhibited an increase in Mg and Ni concentrations relative to grasslands. In greenhouse experiments, Mg toxicity imposed the largest limitation to plant growth on these serpentine soils, expressed most dramatically in savanna soils, where Mg concentrations were as high as 1,575 µg g−1 soil. Plant growth was least constrained on soil from pine woodlands. Pot cultures from grassland soils yielded the greatest AMF species richness and sporulation. Richness and sporulation were reduced in savanna and pine woodland soils, although pine invasion did not alter fungal diversity indices. The negative impact that pines had in the early stages of succession on fungal abundance and plant growth, along with the decreased serpentine signal seen in woodland soils, have important implications for the restoration and management of plant species diversity on these serpentine sites. Altered soil chemistry and depleted AMF populations may be factors that function to limit the success of herbaceous vegetation adapted to serpentine soils.