Negative environmental impacts from mineral fertilisers and pesticides used in conventional cropping have raised concern over the sustainability of arable crop production. Organic cropping uses alternatives that avoid many of these negative environmental effects; however, crop yields can be significantly reduced, possibly due to a lower proportion of plant-available nutrients. To gain insights into the molecular effects of organic compared to conventional cropping systems on plant utilisation of nutrients, we used proteomics to analyse winter wheat (Triticum aestivum). Our aim was to investigate the effects of contrasting fertility management and crop protection regimes in organic and conventional cropping systems on the wheat flag leaf proteome and the association between the proteome and physiological traits. Wheat flag leaves were flash-frozen, lyophilised and milled prior to protein extraction (TCA/acetone) and analysed using 2D gel electrophoresis and MALDI-TOF MS. The abundance of 111 protein spots varied significantly between fertilisation regimes. Flag leaf N and P composition were significant drivers of differences in protein spot abundance, including major proteins involved in nitrogen remobilisation, photosynthesis, metabolism and stress response. These results indicate that molecular-based mechanisms are involved in the effect of contrasting cropping systems on nutrient utilisation and wheat grain yield. Using a functional genomics approach, we were able to identify proteins that are linked to causal genes, enabling the potential development of functional molecular markers for crop improvement in nutrient use efficiency.