Agnoprotein (Agno) is an important regulatory protein of JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) and these viruses are unable to replicate efficiently in the absence of this protein. Recent 3D‐NMR structural data revealed that Agno contains two alpha‐helices (a minor and a major) while the rest of the protein adopts an unstructured conformation (Coric et al., 2017, J Cell Biochem). Previously, release of the JCV Agno from the Agno‐positive cells was reported. Here, we have further mapped the regions of Agno responsible for its release by a structure‐based systematic mutagenesis approach. Results revealed that amino acid residues (Lys22, Lys23, Phe31, Glu34, and Asp38) located either on or adjacent to the hydrophilic surface of the major alpha‐helix domain of Agno play critical roles in release. Additionally, Agno was shown to strongly interact with unidentified components of the cell surface when cells are treated with Agno, suggesting additional novel roles for Agno during the viral infection cycle. JC virus agnoprotein plays critical regulatory roles during the viral replication cycle. Previously, the release of agnoprotein from agnoprotein‐positive cells was reported. In this work, we have further mapped the region(s) responsible for its release. Data showed that the specific amino acid residues located on the hydrophilic surface (designated as release surface) of the major alpha helix domain of agnoprotein, including Lys22, Lys23, Phe31, Glu34, and Asp38 play important roles in this process. Additionally, agnoprotein was found to strongly interact with the unidentified components of the cell surface, suggesting additional roles for it during the viral replication cycle.