The co-product of anaerobic digestion, digestate, is nitrogen (N) rich; however, the forms and accessibility of this N by the crops have not been fully explored. This study aimed to determine the mineralization parameters of digestate N and to assess its availability for annual ryegrass (Lolium multiflorum Lam.). Four digestate rates of 0 (control), 38, 75, and 150 mg N kg^-1 soil (equal to 0, 90, 180, and 360 kg total N ha^-1) were applied to a silty clay loam soil in a completely randomized block design with four replications in a greenhouse study. A 100 d aerobic incubation experiment was also conducted with 0 and 150 mg digestate N kg^-1 rates at 25 °C. Digestate feedstock included cattle manure (28%), hay (15%), and silage corn (Zea mays L.; 57%). Total plant biomass and N uptake increased linearly with digestate application rate with average apparent N recovery of 37%. Potentially mineralizable N (N₀) and mineralizable N rate constant (k) were not significantly different in digestate and control treatments; however, a flush of digestate organic N (30 mg N kg^-1) released right after mixing the digestate with soil. Evidences of N immobilization with digestate application were observed in greenhouse study. Majority of plant-available digestate N was in form of NH₄⁺-N; therefore, NH₄⁺-N can be used for estimation of available digestate N for crops. Results need to be validated for specific feedstock and soil properties under field conditions. Further research is needed to assess how long-term build-up of digestate organic N may impact the N availability for crops.