A more efficient procedure for the extraction of β-D-glucan from Lentinus edodes (Berk.) Sing, (shiitake mushroom) was designed in this study. The β-D-glucan isolated through ethanol precipitation and freeze drying in liquid nitrogen was found to be lentinan. Purity tests carried out using a carbohydrate analysis column, a chromatographic method, has shown a significant lentinan purity of 87.50%. The commercial value of this method is increased further by its being less time consuming, more efficient, and of low cost. Three experiments involving mouse models were designed to evaluate the efficacy of this extract. The pretreatment cohort was to administer this lentinan for 7 days before inoculation with the K36 cells (murine leukemic cell line). In the simultaneous-treatment cohort lentinan was administered together with the inoculation of K36 cells for 7 days. Lastly, in the posttreatment cohort, lentinan was administered 7 days after tumor cells had been inoculated. The highest antitumor activity was observed with the pretreatment cohort. Tumor inhibition rates (TIRs) of 94.44%, 88.59%, and 83.14% were observed with pretreatment, simultaneous-treatment, and posttreatment, respectively. In comparison, a crude L. edodes homogenate showed TIRs of 55.20%, 45.32%, and 43.06%, respectively, for the three experiments. This indicated that extracted lentinan was much more effective in the prevention of tumor formation. Ultrastructural studies revealed a drastic reduction in the number of retroviruses in the induced tumor and most of those were defective. In addition, significant induction of interferon-γ, tumor necrosis factor-α, and interleukin-2 was observed in the mouse model. Thus, host T-cell stimulations by lentinan could well have a role in the antitumor activity observed.