We have studied the influence of long-term fertilization on organic C, nutrients, microbial biomass of soil, and grain yield of rice (Oryza sativa L.) after 18 years of inorganic and organic fertilizer application to an infertile paddy field under subtropical conditions. After 18 years of fertilization, soil organic C and total N contents were 9.56~12.17 and 1.01~1.25 g kg−1, which were 190~269% and 135~191% higher than those of the initial soil, respectively. Organic manure application increased soil organic C, total N, available N, and available P contents by 19.2%, 14.4%, 13.2%, and 78.3% on average compared with organic manure-omitted treatments. Phosphorus fertilizer application increased soil available P content more significantly than organic manure application, and it was an average 385% higher than P-omitted treatments. Soil microbial biomass C, N, and rice yield were 48.9%, 33.2%, and 133% higher in organic manure application treatments and 36.8%, 38.8%, and 239% higher in P fertilizer application treatments than organic manure-omitted or P-omitted treatments, respectively. Incorporation of organic manure-enhanced and P fertilizer-enhanced rice yields by 382% compared with the unfertilized treatment and yield increment index was 123% compared with that observed during 1991–1994, implying that organic manure application combined with P fertilizer was needed to ensure high and sustainable productivity. Rice yield was significantly correlated with all soil chemical properties except available K content, and stepwise regression analysis showed that soil available P content was the limiting factor to rice yield.