Lymphoid organs play an important role in prion disease development and progression. While the role of lymphoid organs and changes in immune-related genes have been extensively investigated in scrapie-infected animals, innate immunity has not. Previous studies examined lymphocyte function in scrapie-infected C3H/HeJ mice, which exhibit defects in lipopolysaccharide (LPS) response now known to result from a mutation in Toll-like receptor (TLR) 4. We examined immune function in scrapie-infected CD1 mice, which are LPS responders. Lymphocyte proliferation from CD1 mice infected with either 139A or ME7 scrapie was measured in response to concanavalin (Con) A or LPS at 1 and 3 months after infection. Following LPS exposure, mice infected 3 months with ME7, but not 139A, demonstrated significantly decreased lymphocyte proliferation compared to controls. After Con A exposure, lymphocyte proliferation in scrapie-infected mice did not differ from controls. Gender-specific comparison of lymphocyte proliferation showed significant decreases in mitogenic responses in females infected 3 months with either 139A or ME7, compared to controls. Males infected for 3 months with ME7, but not 139A, showed significantly decreased proliferation after lymphocyte exposure to LPS, but not Con A. Neither gender showed changes in lymphocyte proliferation after 1 month of scrapie infection. Innate immune activation of peritoneal macrophages was determined via production of nitric oxide (NO), IL-6, and TNF-α after exposure to TLR ligands. TNF-α and IL-6 production were reduced in macrophages from females infected with either scrapie strain for 3 months, while NO production after TLR agonist plus IFN-γ exposure was decreased in both females and males infected for 3 months with 139A, compared to ME7. These data demonstrated altered innate immunity, suggesting hormonal and/or other gender-specific regulation may contribute to gender differences in some immune functions. Our data demonstrate lymphocyte proliferation and innate immune functioning in scrapie-infected mice deteriorate with disease progression.