Dengue virus (DENV) is an arbovirus that causes infections ranging from mild to severe disease in humans. DENV encodes non-structural protein 1 (NS1), which acts as a pathogen-associated molecular pattern by activating Toll-like receptor 4 (TLR4). Given the previously reported detection of DENV in the semen and the abundant expression of TLR4 in the epididymis, we hypothesized that DENV may trigger epididymitis through NS1-mediated TLR4 activation. Initial segment and cauda epididymidis from naïve C57BL/6 mice were cultured ex vivo with recombinant NS1 from DENV serotype 2 (DENV-2 NS1; 20 µg/mL; 3 h) to evaluate the expression of cytokines and chemokines. Ultrapure LPS from E. coli (50 ng/mL) and sterile saline were used as controls. DENV-2 NS1 induced cytokine/chemokine responses characterized by region-specific patterns of transcript upregulation: Tnf, Il1b, Ifnb1, Ifng, Ccl3, and Ccl5 in the initial segment, and Il6, Tnf, Il1b, Il10, Ifnb1, Ifng, Ccl3, Ccl4, Ccl5, and Cxcl2 in the cauda epididymidis. These responses were similar to those induced by LPS in both epididymal regions and were partially blunted by the TLR4 antagonist CLI-095. To determine whether similar responses occur in vivo, male BALB/c mice were challenged intracerebrally with DENV-2 NGC (40 X LD₅₀); mock controls received 199 medium (vehicle). The initial segment, caput, corpus, and cauda epididymidis were harvested 1 to 7 days post-infection (dpi). DENV-2 NGC infection induced time-dependent upregulation of Ifnb1, Ifng, and Tnf in the initial segment (1, 3, and 7 dpi), caput (1 and 3 dpi), and corpus epididymidis (1, 3, 5, and 7 dpi). Only Ifnb1 and Tnf were upregulated in the cauda epididymidis (1, 3, and 5 dpi). These findings indicate that DENV-2 can trigger epididymitis, in part through NS1-induced TLR4 activation, and suggest a potential impact of DENV-2 infection on male fertility. Support: FAPESP (2021/04746-3; 2025/04157-9). Ethics approvals No. 2132170322; L-022/2019-A1.