Nanomedicine-based tumor-targeted therapy has emerged as a promising strategy to overcome the lack of specificity of conventional chemotherapeutic agents. "Passive" targeting caused by the tumor EPR effect and "active" targeting endowed by the tumor-targeting moieties provide promising biomedical utilities and cancer therapy strategies for nanomedicine. However, as the nanoparticles are exposed to biological fluids, a large number of protein molecules will be adsorbed on their surface, known as protein corona, which may alter the targeting ability of the nanoparticles. The impact of different protein corona on the "passive" and "active" targeting behaviors is still ambiguous. Herein, three kinds of aqueous soluble Fe3O4 nanoparticles with different surface modifications were synthesized and applied to explore the correlation between their protein corona and passive/active tumor-targeting abilities. In the in vitro and in vivo studies, the protein corona exhibited completely different effects on the active and passive cancer-targeting capability of the particles. The particles presented active cancer-targeting ability if there was enough interaction time between the particles and cells. This was mainly due to the dynamic evolution of the protein corona, the proteins of which may be outcompeted by the cancer cell membrane and determine the targeting abilities. Unfortunately, the protein corona also inevitably accelerated RES/MPS uptake after the particles were injected into the body, which almost completely disabled the active targeting abilities of the particles. We believe that this in-depth understanding of protein corona will provide new ideas on the tumor-targeting mechanisms of nanoparticles and present a feasible approach to designing targeted drugs in the future.