The application of 33P or 32P isotopes to directly trace phosphorus (P) uptake during arbuscular mycorrhizal (AM) symbiosis is limited by the radioactivity of the two P isotopes, especially under field conditions. A potential alternative method for tracing P uptake in plant-soil systems relies on the analysis of the stable oxygen (O) isotopes of ortho-phosphate (Pi); however, little is known about the fate of the P–O bond during Pi uptake in AM symbioses. This study investigated whether the abundance of 18O in Pi extracted from the shoots of maize increased after 18O-labeled Pi added to soil was taken up by either roots of maize or AM extraradical hyphae. A two-compartment culture system, consisting of a root and AM hyphal compartment (RHC, including both roots and AM hyphae) and an AM hyphal compartment (HC, including only hyphae) was designed, and the AM fungus Funneliformis mosseae was used to inoculate the roots of maize. Our results indicated that the abundance of 18O in Pi extracted from the maize shoots increased significantly 3 months after the addition of 18O-labeled Pi to the soil in the pots which only contained roots. The abundance of 18O was much lower than expected, however, which suggests a great majority of 18O in labeled Pi was lost in the soil or during Pi metabolism in the shoots of maize. The abundance of 18O in Pi extracted from the maize shoots did not increase 3 months after 18O-labeled Pi was added to the HC, and therefore, loss of 18O in labeled Pi may also occur during Pi metabolism in AM hyphae. Use of 18O-labeled Pi as a qualitative tracer of P uptake during AM symbiosis appears unfeasible for such a long-term (3 months) experiment, although it should be investigated in a short-term labeling experiment.