To investigate the optimal timing for initiating anticancer immunotherapy with the TLR4 agonist EC-LPS plus the TLR9 agonist CpG, mice were injected i.v. with B16-F10 melanoma cells, and the TLR4/TLR9 agonist complex was injected i.p. either before (prophylactic) or after (therapeutic) tumor cell inoculation every three days for three doses. Here, TLR4 is linked to melanoma.