Buckwheat (Fagopyrum esculentum) is consumed as a pseudocereal in Asia and Western countries, and is recognized as a functional food. However, several proteins from buckwheat have strong allergenicity and cause severe symptoms, such as an anaphylaxis, in hypersensitive patients. Here, we engineered strains of Lactococcus lactis NZ9000 (NZ9000) that express Fagag1, a major allergenic storage protein of buckwheat, fused with or without green fluorescent protein (GFP). Codon-optimized sequences of Fagag1 and GFP-Fagag1 were individually cloned into the L. lactis expression vector pNSH, containing a nisin-inducible promoter and 6x histidine-tag sequence. The resulting plasmids were separately introduced into NZ9000, and nisin-dependent expression of recombinant Fagag1 (rFagag1) and rGFP-Fagag1 was confirmed by Western blotting and confocal laser scanning microscopy. rFagag1 and rGFP-Fagag1 were individually purified by immobilized metal-ion affinity chromatography techniques, and the allergenicity of the purified proteins was then evaluated in in vitro cultures of splenocytes isolated from buckwheat crude protein-immunized mice. Treatment of splenocytes with rFagag1 markedly induced the mRNA expression of interleukin (IL)-4, IL-13, and IL-17F, which are known mediators of allergic inflammation. Similar expression levels of IL-4 and IL-17F mRNA were observed in the splenocytes stimulated with purified rGFP-Fagag1; however, the increases from baseline were significantly lower than those observed in rFagag1-treated cells. Recent evidence suggests that the mucosal (i.e., oral or intranasal) application of lactic acid bacteria genetically modified to produce allergen is a promising strategy for allergy therapy. Therefore, our present results suggest that NZ9000 expressing immunoreactive rFagag1 or rGFP-Fagag1 may be a powerful candidate for the prevention and therapy of buckwheat allergy. However, the future application of prophylactic and therapeutic strategies based on NZ9000 strains first requires a clear demonstration of efficacy in in vivo trials.