Long-term application of manure can lead to phosphorus accumulation in the soil and increase the risk of P delivery to tile drainage waters. A long-term study (1998–2009) was conducted on eight tile-drained field plots, ranging in size from 0.19 to 0.47 ha, to investigate the effects of long-term surface application of poultry manure on soil phosphorus dynamics and PO4-P loss in tile drainage in Iowa under a corn-soybean rotation system. The experimental treatments included two poultry manure treatments, applied on an N basis at target rates of 168 kg N ha−1 (PM) and 336 kg N ha−1 (PM2), each with three replications, and one chemical fertilizer treatment of urea ammonium nitrate (UAN) at a rate of 168 kg N ha−1 with two replications. Actual manure application rates and estimated plant available N (PAN) varied annually. Bray1-P methods were used to analyze deep core soil samples at five depths (0–15, 15–30, 30–60, 60–90, and 90–120 cm), which were collected in the spring and fall on the half of each plot planted to corn. Tile drainage samples were collected from each actively draining plot equipped throughout the spring and fall drainage season each year, and PO4-P concentrations were analyzed. The results of this study indicated PM2 and PM resulted in a statistically significant increase in topsoil P at 0–30 cm, with no significant movement of P to deeper soil depths. Although an increase in topsoil P levels was observed with poultry manure application, average annual tile drainage P concentrations were not statistically different throughout the study with average PO4-P concentrations of 0.019 ppm with PM2 application and 0.011 and 0.012 ppm for PM and UAN, respectively. Poultry manure application at agronomical recommended application rates may be an environmentally sound fertilizer option for tile drained fine-loamy, calcareous soils under a corn-soybean rotation. However, surface runoff was not evaluated in this study, and high P levels in topsoil could contribute to elevated PO4-P runoff losses.