Background and aims <p>Arbuscular mycorrhizal fungi (AMF) play an essential role in plant nutrition in both natural and agro-ecosystems. However, how soil nutrient availability simultaneously regulates AMF diversity and contribution to plant nutrition, requires more attention. We hypothesised that the interaction between the availability of key soil macronutrients phosphorus (P) and nitrogen (N) regulates AMF contribution to wheat nutrition and that nutrient availability will simultaneously influence AMF community composition.</p> Methods <p>We tested this using a unique long-term P fertilisation trial, sampling wheat roots across two growing seasons. Expression of wheat mycorrhizal nutrient transporters was quantified by RT-qPCR and AMF communities were characterised by ITS2 metabarcoding. Complementary experiments under controlled conditions examined how the interaction between P and N regulates arbuscular mycorrhizal function in plant nutrition.</p> Results <p>Field-grown wheat showed campaign-specific effects of P fertilisation on AMF colonisation and nutrient transporter expression, which coincided with shifts in plant N status. Controlled experiments confirmed that colonisation depends on the limitation of either P or N, but that the regulation of peri-arbuscular phosphate, ammonium and nitrate transporters depended on the limiting nutrient. AMF communities also responded to soil P availability, with the genus <i>Funneliformis</i> consistently dominating under high P conditions.</p> Conclusions <p>Our findings demonstrate that P and N availability jointly shape root AMF communities and regulate their nutritive function in wheat. The combination of community profiling and mycorrhizal molecular markers provides a valuable approach for understanding the AMF contribution to plant nutrition across agroecosystems, and therefore can be used for optimising agroecological practices.</p>