Soy isoflavones may have applications in cancer prevention and anti-inflammation, therefore this study was conducted to investigate the effect of dietary supplementation with black soybean on the immune response in the senescence-accelerated-prone mice (SAMP8) and -resistant mice (SAMPR1, as controls). The mechanism of isoflavones was also investigated. Six-month-old male SAMP8 and SAMR1 mice were divided into the control groups and experimental groups supplemented with nanonized (Nano-soy) or microparticled (Micro-soy) black soybeans (n = 8/group), respectively for 12 weeks. Human peripheral blood mononuclear cells (PBMC) and murine splenocytes were stimulated with mitogens and cytokines were determined by reverse transcriptase-polymerase chain reaction and/or ELISA. The results showed that body weight, food intake, and relative weights of organs did not differ among the SAMP8 control and experimental groups. Isoflavone (daidzin and genistin) intake was higher in the Nano-soy group than the Micro-soy group. The lymphoproliferation and production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in the Nano-soy group had a significantly higher (P < 0.05) than those in the control and Micro-soy groups. The Nano-soy supplemented mice reached these cytokine levels similar to SAMR1 mice. This result was consistent with the in vitro data that daidzein (a metabolite of daidzin), at a concentration of 10 muM, increased IL-2, IL-4, and IFN-gamma production from phytohemagglutinin-stimulated PBMC (P < 0.05). However at higher concentrations (> 50 microM), daidzein only reduced IL-10 and IFN-gamma levels, whereas genistein reduced levels of the IL-2, IL-4, IL-10, IFN-gamma mRNA and protein and these results suggest that the Nano-soy supplementation improved immune response in SAMP8 mice which may be attributable to higher daidzin content in the black soybean preparation.