Prenatal alcohol exposure (PAE) leads to immune system dysfunction and increased susceptibility to autoimmune diseases. To explore potential mechanisms by which PAE induces neuroimmune dysfunction, the current study examined the impact of PAE on toll-like receptor 4 (TLR4) and TLR7/8 activation. Pregnant rats were divided into two groups: PAE - liquid ethanol diet ad libitum, and CON - pelleted control diet ad libitum. Male and female offspring were challenged in adulthood with LPS (40 µg/kg; activates TLR4), R848 (1 mg/kg; activates TLR7/8), or DMSO (vehicle), followed by brain sample collection to measure cytokine levels (IL-1β, IL-10, IL-13, IFN-γ, IL-4, IL-5, IL-6, KC/GRO, TNF-α). Analysis of brain cytokines indicates that differential PAE responses to LPS or R848 occur mainly 90 minutes after injection and in a sex-dependent manner. PAE females, when challenged with R848, exhibited higher levels of IL-6 in the amygdala compared to CON and LPS. Additionally, PAE males displayed a greater IL-5 and IL-13 response to R848 in the amygdala compared to CON and LPS males. Also in the amygdala, LPS increased IL-6 levels in PAE but not CON males. Moreover, PAE males challenged with LPS showed increased IFN-γ, IL-5, and IL-10 levels compared to the CON group in the hypothalamus. No significant effects of PAE were observed in the hypothalamus of females. Collectively, these findings indicate that prenatal alcohol exposure (PAE) leads to heightened responsiveness to immune challenges and elevated cytokine production, thereby playing a crucial role in the development of autoimmune inflammatory diseases.