Background <p>Breast cancer (BC) progression is influenced by both pro- and anti-inflammatory cytokines. Interleukin (IL)-12 and IL-35 play opposing immunological roles, functioning as pro- and anti-inflammatory cytokines with anti- and pro-tumorigenic effects, respectively. Resiquimod (RSQ), a Toll-like receptor 7/8 (TLR7/8) agonist, is an immunomodulatory agent that enhances antitumor immune responses. However, the influence of RSQ on the balance between pro- and anti-inflammatory cytokines in BC patients remains poorly understood.</p> Aim <p>This study aimed to assess the effect of RSQ on the IL-12/IL-35 balance in patients with BC.</p> Subjects and methods <p>The study included 26 female patients with invasive non-metastatic BC and 20 age-matched healthy female controls. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured in the presence or absence of RSQ. Levels of IL-12 and IL-35 in culture supernatants were quantified using sandwich enzyme-linked immunosorbent assay (ELISA) kits.</p> Results <p>Baseline IL-35 levels were significantly higher in BC patients than in healthy controls, resulting in a significantly lower baseline IL-12/IL-35 ratio in the patient group. RSQ stimulation significantly increased IL-12 levels while significantly decreasing IL-35 levels in both groups. Consequently, the IL-12/IL-35 ratio increased significantly in both BC patients and healthy controls, with a more pronounced shift observed in the control group. Notably, RSQ-stimulated IL-35 levels remained significantly higher in BC patients compared with controls.</p> Conclusion <p>These findings indicate a predominance of immunosuppressive activity in PBMCs from BC patients, largely driven by elevated IL-35 levels. Resiquimod may exert anti-tumorigenic effects in BC by shifting the IL-12/IL-35 balance toward a pro-inflammatory immune profile.</p>