Diffuse large B-cell lymphoma (DLBCL) is the most common hematological malignancy in humans and dogs. Several studies disclosed some similarities between the two species, including the constitutive activation of the NF-κB pathway as a fundamental underlying pathogenetic mechanism. In humans, the downregulation of IL-1R8 is implicated in DLBCL development, but its role in dogs has not been explored so far. To gain insight into the pathogenesis of this tumor in dogs, we evaluated the mRNA and protein expression of IL-1R8 in 12 control lymph nodes obtained from dogs not bearing tumors and from 50 dogs with DLBCL. Moreover, we analyzed through qRT-PCR the expression of TLR7, TLR9, MYC, and p52 genes that are known to be involved in the IL-1R8 regulatory network. IL-1R8 and p52 were downregulated in DLBCLs compared to control lymph nodes (p < 0.001), while a higher expression of TLR7, TLR9, and MYC was observed in DLBCLs (p < 0.01). Immunohistochemistry confirmed the gene expression results, revealing a significantly lower IL-1R8 staining score in DLBCLs compared to control lymph nodes (p < 0.0001). Taken together, these results suggest that IL-1R8 downregulation may represent one of the mechanisms driving DLBCL pathogenesis in dogs, mainly through the dysregulation of the Toll-like/interleukin receptors signaling cascade and the aberrant activation of the classical NF-κB pathway.