A long-term field experiment was conducted at the Ecological Experimental Station of Red Soil, Chinese Academy of Sciences in order to investigate rehabilitation of fertility in infertile red soils with application of fertilizers. This paper reported mainly results of the experiment about changes in soil biochemical properties. Application of organic manure at a rate of 4500~9000kg/hm² for 5 years increased organic carbon content by 2.1~7.5g/kg and total nitrogen by 0.18~0.71g/kg in surface 20cm of infertile red soils, while application of inorganic fertilizers only by 2.0g/kg and 0.22g/kg, respectively. When the application rate of organic manure was 9000kg/hm², 5 years' application could increase the organic carbon content from 1.3~3.0g/kg to 7.0~9.7g/kg and the total nitrogen from 0.34~0.41g/kg to 0.70~1.05g/kg in surface 20cm of infertile red soils, which were close to the level of normal upland red soil. Organic matter accumulation also varied with the parent material, landuse pattern and organic manure application rate. Application of organic manure, moreover, significantly increased the content and ratio of organic carbon in macroaggregates, enzyme activity, microorganism population, indicating its important role for improving nutrient availability and organic matter quality.