LiFePO4 has attracted broad attention as a promising cathode material for lithium ion batteries. One of the key issues related to LiFePO4 performance lies on the intrinsic characteristic of Li/Fe inter-site mixing. To explore the effect of the defect chemistry on electrochemical behavior, LiFePO4 is synthesized by hydrothermal method with pH value varying from 11.04 to 5.40. The results show that pure phase of LiFePO4 could only be obtained at slightly basic and neutral conditions, and Rietveld refinements reveal that the degree of vacancies and inter-site mixing increase with decreasing pH value. The amounts of Fe on Li sites is nearly zero at pH value of 8.19, whereas 3.5% at 6.30. EIS measurements confirm that the occupation of Fe on Li sites will block the one-dimensional tunnel for lithium ion diffusion. It is vital to prevent the defect chemistry of LiFePO4 by optimizing the synthesis conditions.