The use of bovine metalloproteinases and IFNγ to hasten mammary gland involution and the stimulation of immune system response has been proposed to accelerate the dry off period and diminish the risk of intramammary infections. The protein nanoparticles technology has shown to be an economically viable production strategy of recombinant proteins. The objective of this work was to study the activity of bovine metalloproteinase 2 and 9 (MMP-2, MMP-9) and IFNγ produced in Lactococcus lactis either as nanoparticles (Lu et al. Mol. Biotechnol. 2013 54:170–176) or soluble recombinant proteins. The activity of IFNγ was tested using a macrophage activation test along with an assay of induction of nuclear factor kappa-β (NF-κβ) expression in mammary epithelial cells. Monocytes were isolated from cow blood, differentiated to macrophages, and treated with IFNγ for 24 h at 37°C, 5% CO₂ while primary culture of mammary gland epithelial cells were treated with IFNγ for 4 h at 37°C, 5% CO₂. Cell samples were taken to analyze the expression of NF-κβ by qPCR. Activity of MMP-2 and MMP-9 soluble or as nanoparticles was tested by both zymography and ex vivo. For the ex vivo assay, mammary gland explants were incubated with MMP for 7 h at 37°C and 5% CO₂. Then, apoptotic and proliferation gene expression was assessed by qPCR (PCNA, SPC25, CASP3 and BNIP). The sensitivity of NF-κβ assay was greater than the macrophage activity test (detection limit of 19.6 µg and 140 µg IFNγ, respectively; P < 0.05). Soluble IFNγ upregulated (P < 0.05) NF-κβ, whereas similar amounts of IFNγ nanoparticles had no significant effect. Metalloproteinase activity was detected for MMP-2 and MMP-9 in both forms by zymography. As expected, the soluble form was more active than the nanostructured form (P < 0.05). Only soluble MMP9 activity was detected ex vivo, stimulating (P < 0.001) the expression of SPC25 and PCNA proliferative genes. In conclusion, the bovine recombinant MMP and IFNγ proteins elicited the expected biological response and thus they can be used in further in vivo dry-off studies in dairy cows.