The effect of phosphate treatment on lead relative bioavailability (Pb RBA) was assessed in three distinct Pb-contaminated soils. Phosphoric acid (PA) or rock phosphate were added to smelter (PP2), nonferrous slag (SH15), and shooting range (SR01) impacted soils at a P:Pb molar ratio of 5:1. In all of the phosphate amended soils, Pb RBA decreased compared to that in untreated soils when assessed using an in vivo mouse model. Treatment effect ratios (i.e., the ratio of Pb RBA in treated soil divided by Pb RBA in untreated soil) ranged from 0.39 to 0.67, 0.48 to 0.90, and 0.03 to 0.19 for PP2, SH15, and SR01, respectively. The decrease in Pb RBA following phosphate amendment was attributed to the formation of poorly soluble Pb phosphates (i.e., chloropyromorphite, hydroxypyromorphite, and Pb phosphate) that were identified by X-ray absorption spectroscopy (XAS). However, a similar decrease in Pb RBA was also observed in untreated soils following the sequential gavage of phosphate amendments. This suggests that in vivo processes may also facilitate the formation of poorly soluble Pb phosphates, which decreases Pb absorption. Furthermore, XAS analysis of PA-treated PP2 indicated further in vivo changes in Pb speciation as it moved through the gastrointestinal tract, which resulted in the transformation of hydroxypyromorphite to chloropyromorphite.