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The vadose zone is the first natural layer preventing groundwater pollution. Understanding virus transport and retention in the vadose zone is necessary. The effects of different interfaces and mechanisms on virus transport and retention were investigated by studying Escherichia coli phage migration in laboratory-scale columns under unsaturated conditions. The E. coli phage was used as a model virus. Colloid filtration theory, extended Derjagin–Landau–Verwey–Overbeek theory and two−site kinetic deposition model were used to calculate fitted parameters and interaction energies to assess virus retention at different interfaces. The collector diameters and the size of E. coli phages in the influent and effluent were compared to assess the effect of straining. The results indicated that the roles of solid–water interfaces (SWIs) and air–water interfaces (AWIs) in retaining E. coli phages are strongly controlled by the moisture content and hydrochemical conditions. Decreasing the moisture content and increasing the ionic strength (IS) of the suspension increased E. coli phage retention. At suspension ISs of 0.01 or 0.03 M and various moisture contents, E. coli phages were mainly retained at the SWIs rather than AWIs. When the IS was increased to 0.06 M, the viruses were strongly retained by becoming attached to both SWIs and AWIs. The role of straining in virus retention could not be ignored. Viruses were retained more at the SWIs and less straining occurred under acidic conditions than under neutral or alkaline conditions. This was mainly because of the effects of the pH and IS on surface charges and the model virus particle size. This study has important implications for modeling and predicting virus transport in soil affected by rainfall, snowmelt, and human activities (e.g., irrigation and artificial groundwater recharging).