Cancer therapeutic nanovaccines are ideal tools to inhibit tumor growth and provide the body with continuous protecting immune surveillance.However,the conventional subcutaneous (SC) vaccination normally induces limited anti-tumor immune responses with low therapeutic efficacy.Herein,we devised clay-based nanovaccines and directly delivered them to the spleen via intravenous (Ⅳ) injection to induce the stronger anti-tumor immunity with higher efficacy for tumor prevention and treatment.The clay,i.e.,layered double hydroxide (LDH) was prepared as nanoadjuvant with the average size from 77 to 285 nm and co-loaded with the model antigen ovalbumin (OVA) and bioadjuvant CpG to form CpG/OVA-LDH (CO-LDH) nanovaccines.We found that CO-LDH-215 (the size of LDH was 215 nm) promoted dendritic cells to present the most antigen,and moreover showed the highest spleen enrichment (～ 1.67％ of CO-LDH-215 endched in the spleen at 24 h post Ⅳ injection).The in vivo immunologic data showed that CO-LDH-215 induced the most potent anti-tumor immune responses and completely prevented the growth of E.G7-OVA tumor in the mouse model.Furthermore,Ⅳ injected CO-LDH-215 nanovaccine more effectively delayed tumor growth than that SC injected,largely due to the direct and quick delivery of more nanovaccines to the spleen.This study demonstrates that the therapeutic efficacy of nanovaccines can be greatly enhanced by targeted delivery of nanovaccines to the spleen via the proper vaccination route.