Real-time imaging of single virus particles allows the visualization of subtle dynamic events of virus-host interaction. During the human immunodeficiency virus (HIV) infection of resting CD4 T lymphocytes, overcoming cortical actin restriction is an essential step, but the dynamic process and mechanism remain to be characterized. Herein, by using quantum dot (QD) encapsulated fluorescent viral particles and single-virus tracking, we explored detailed scenarios of HIV dynamic entry and crossing the cortical actin barrier. The fine-scale temporal and spatial processes of single HIV virion interaction with the cortical actin were studied in depth during virus entry via plasma membrane fusion. Individual HIV virions modulate the subtle rearrangement of the cortical actin barrier to open a door to facilitate viral entry. The actin-binding protein, α-actinin, was found to be critical for actin dynamics during HIV entry. An α-actinin-derived peptide, actin-binding site 1 peptide (ABS1p), was developed to block HIV infection. Our findings reveal an α-actinin-mediated dynamic cortical actin rearrangement for HIV entry, and identify an antiviral target as well as a corresponding peptide inhibitor based on HIV interaction with the actin cytoskeleton.