Abstract: Adopting proper management practices for poultry litter (PL) is critical to increase N efficiency, maximize yield, and reduce negative environmental impacts. This study investigated the effects of application time (fall and spring) of two PL rates (9.0 and 18.0 Mg ha−1) and commercial N-P-K fertilizer blend (224 kg N ha−1, 84 kg P ha−1, 145.6 kg K ha−1) on selected soil properties and corn (Zea mays L.) grain yield in Central Kentucky. Application time response was measured by grain yield, whole plant nutrient concentrations and uptake, and soil nutrients availabilities. Application time of the PL and commercial fertilizer did not influence grain yield or plant nutrient concentrations and uptake. Grain yield was increased by the two PL rates, and no difference was found among the PL and commercial fertilizer. Whole plant uptake and concentrations of N, P, and K increased when PL plant available N-P-K increased. Soil inorganic N was 17% (18 kg ha−1) greater from spring than fall applications of the high PL rate when sampled midseason. Application time did not influence inorganic N at the low PL rate. Greater soil available K resulted from fall than spring applications of the PL, but Mehlich 3 available P was not influenced by PL application time. Results indicate that fall applications of PL have equal potential as spring applications without compromising plant available nutrients and yield. In addition, the low PL rate was adequate in N, P, and K to meet the soil’s requirement for optimum yield, without significant nutrient accumulation and loss.