Phosphorus (P) is a nonrenewable resource, which is one of the major challenges for sustainable agriculture. Although phosphite (Phi) can be absorbed by the plant cells through the Pi transporters, it cannot be metabolized by plant and unable to use as P fertilizers for crops. However, transgenic plants that overexpressed phosphite dehydrogenase (PtxD) from bacteria can utilize phosphite as the sole P source. In this study, we aimed to improve the catalytic efficiency of PtxD from sp.4506 (PtxD), by directed evolution. Five mutations were generated by saturation mutagenesis at the 139th site of PtxD and showed higher catalytic efficiency than native PtxD. The PtxD showed the highest catalytic efficiency (5.83-fold as compared to PtxD) contributed by the 41.1% decrease in the and 2.5-fold increase in the values. Overexpression of PtxD in and rice showed increased efficiency of phosphite utilization and excellent development when phosphite was used as the primary source of P. High-efficiency PtxD transgenic plant is an essential prerequisite for future agricultural production using phosphite as P fertilizers.