The present study analyses the effect of flax-seed oil rich in n-3 polyunsaturated fatty acids (PUFAs), the probiotic strain Lactobacillus plantarum - Biocenol™ LP96 and their combination on the expression level of selected Toll-like receptor (TLR) genes (TLR2, TLR4, TLR5, TLR9) and their downstream molecules (myeloid differentiation factor 88, MyD88; nuclear factor-κB, NF-κB) in the jejunum of gnotobiotic pigs challenged with enterotoxigenic Escherichia coli (ETEC). The results show that both immunomodulators are able to modulate the RNA level of at least one of the target molecules and thus regulate pathogeninduced inflammation. We confirmed that not only probiotic lactobacilli or flaxseed oil alone but also their synergistic action has great potential in the prevention and treatment of porcine colibacillosis. The results give an insight into one of the possible mechanisms by which natural agents, such as probiotic lactobacilli and flax-seed oil, exert their immunoregulatory properties during pathogen-induced inflammation.