VP60 capsid protein is the major structural and immunogenicity protein of RHDV (Rabbit hemorrhagic disease virus, RHDV), and has been implicated as a main protein antigen in RHDV diagnosis and vaccine design. In this report, egg yolk antibody (IgY) against N-terminal of VP60 was evaluated and developed as a new strategy for RHDV therapy. Briefly, N-terminal of VP60 (∼250aa) fragment was cloned and inserted into pET28a expression vector, and then the resultant plasmid, pET28a/VP60-N, was transformed into E. coli BL21(DE3) for recombinant VP60-N protein (rVP60-N) expression. Next, the rVP60-N was purified by Ni(+)-affinity purification chromatography and identified by Western blotting with RHDV antiserum. After immunizing the chickens with rVP60-N, the anti-rVP60-N IgY was isolated, and the activity and specificity of the IgY antibody were analyzed by ELISA and Western blotting. In our results, the rVP60-N could be expressed in E. coli as soluble fraction, and the isolated anti-rVP60-N IgY demonstrated a high specificity and titer (1:22,000) against rVP60-N antigen. For further evaluation of the IgY efficacy in vivo, rabbits were grouped randomly and challenged with RHDV, and the results showed that anti-rVP60-N IgY could significantly protect rabbits from virus infection and promote the host survival after a sustained treatment with anti-rVP60-N IgY for 5 days. Taken together, our study demonstrates evidence that production of IgY against VP60 could be as a novel strategy for the RHDV therapy.