One-shot medical image segmentation faces fundamental challenges in prototype representation due to limited annotated data and significant anatomical variability across patients. Traditional prototype-based methods rely on deterministic averaging of support features, creating brittle representations that fail to capture intra-class diversity essential for robust generalization. This work introduces Diffusion Prototype Learning (DPL), a novel framework that reformulates prototype construction through diffusion-based feature space exploration. DPL models one-shot prototypes as learnable probability distributions, enabling controlled generation of diverse yet semantically coherent prototype variants from minimal labeled data. The framework operates through three core innovations: (1) a diffusion-based prototype enhancement module that transforms single support prototypes into diverse variant sets via forward-reverse diffusion processes, (2) a spatial-aware conditioning mechanism that leverages geometric properties derived from prototype feature statistics, and (3) a conservative fusion strategy that preserves prototype fidelity while maximizing representational diversity. DPL ensures training-inference consistency by using the same diffusion enhancement and fusion pipeline in both phases. This process generates enhanced prototypes that serve as the final representations for similarity calculations, while the diffusion process itself acts as a regularizer. Extensive experiments on abdominal MRI and CT datasets demonstrate significant improvements respectively, establishing new state-of-the-art performance in one-shot medical image segmentation.