On-farm anaerobic treatment is used for solids, odor and pathogen removal, and energy recovery. Not much information is available on the effect of this treatment method on phosphorus (P) dynamics. The objective of this study was to investigate the effect of different inoculum-to-substrate ratios (ISR) on P extractability from anaerobically digested dairy manure. Dairy manure (substrate) and anaerobically digested dairy manure (inoculum) were collected from a full-scale on-farm anaerobic digester. High ISR (HIS, with ISR = 2.0) and low ISR (LIS, with ISR = 0.3) samples were prepared on a volatile solids (VS) basis. These samples were subjected to mesophilic (35°C) anaerobic digestion in batch reactors without mixing for 120 d. Specific methane yield, TS, and VS removal were higher in the LIS system compared to the HIS system. Serial and repeated extraction methods were used to characterize P extractability. De-ionized (DI) water and MgCl2 were used as extractants in the serial extraction method with extractant-to-manure ratios (EMR) ranging from 3 to 127. For repeated extraction, manure was extracted six times with DI water (EMR = 3). While anaerobic treatment decreased water-extractable P (WEP) in LIS extracts at EMR values of 3 and 7 by 28% and 24%, respectively, a significant increase (40%) in WEP occurred at an EMR of 3 at HIS. Water-extractable P in all other serial extracts of treated manure was higher than that in extracts of untreated manure regardless of ISR and EMR. Extractability of P increased sharply up to the third extraction step, after which it leveled off. The effectiveness of anaerobic digestion at LIS to reduce P extractability was also observed up to the second extraction step. In contrast, the digested manure always produced higher WEP levels for the HIS system. Our results indicate that, depending on the ISR used, anaerobic digestion of dairy manure can increase or decrease manure P extractability and, hence, its availability in runoff. However, it appears that this impact could be more pronounced under conditions where low runoff volumes are generated and for the first few precipitation events after land application of manure.