The gut epithelial barrier is a strategic place to prevent, or at least to limit, parasite dissemination upon oral infection with Toxoplasma gondii. Innate immunity to this pathogen results from delicate interactions involving different components of the infecting agent and the host. We herein aimed to examine the molecular mechanism by which protozoan DNA boosts the production of α-defensin-5 (DEFA-5), the main antimicrobial peptide at the target site of infection. The present study shows that DEFA-5 is rapidly upregulated in intestinal epithelial cells following intracellular Toll-like receptor 9 (TLR9) activation by unmethylated CpG motifs in DNA from T. gondii (CpG-DNA). Concomitantly, CpG-DNA purified from the pathogen markedly increased TLR9 mRNA expression levels in the Caco-2 cell line. We further verified that DEFA-5 production was dependent on interferon-β released from these cells upon treatment with CpG-DNA prepared from tachyzoites. Our results suggest that, in protozoan DNA-stimulated intestinal epithelial cells, the TLR9/interferon-β/DEFA-5 pathway may initiate an innate anti-T. gondii response without the need of parasite invasion. These findings highlight the key role of the gut epithelium in Toxoplasma recognition and amplification of local host defence against this microbe, thereby contributing to gain insight into immunoprotective mechanisms and to improve therapeutic strategies.