Nano-hydroxyapatite (NHAP), possessing high defluoridation capacity, has been widely used to remove fluoride (F) from polluted water, but little is known about how it affects the bioavailability and toxicity of soil F towards plants. Here, the impact of NHAP (2% w/w) amendment on immobilization, speciation and accumulation of F was studied in a soil-plant system. The results revealed that the NHAP amendment worked effectively to reduce levels of water-soluble F (37.3%−87.8%) and increase available P (76.6%–147%). X-ray photoelectron spectroscopy analysis indicated that the formation of insoluble CaF2 and the ion exchange of F− with OH− into NHAP might be involved in the mechanism of F immobilization and soil pH elevation. Exposure to NHAP significantly decreased the abundance of Cyanobacteria in tested soils, and Gemmatimonadetes abundance in bulk soil was significantly higher than that in rhizosphere soil at 1,000 mg kg−1 F spiked level. Additionally, NHAP amendment decreased F accumulation in wheat shoots (9.10%–18.7%) and roots (3.88%–22.4%), which could mainly be attributed to the reduction of soil bioavailable F and the supplement of Ca from NHAP. These results suggest that NHAP could be a promising amendment to be applied to acidic soil contaminated with F.