The interactions between HIV-1 and the host cell have been extensively studied, but cell-wide organellar changes caused by the virus have not been defined. Here, we aimed to detail perturbations to the host cell spatial proteome following HIV protein expression. Subcellular fractionation followed by mass spectrometric analysis of Jurkat T cells, in which HIV-expression was uniformly induced, identified thousands of cytoplasmic and membrane-bound proteins and enabled their placement within a multi-dimensional analytic space based on the 7 fractions. Spatial proteomic data were analyzed via a support vector machine bagging classifier to assess the movement of proteins between subcellular compartments. The proteins were also examined for absolute movement within 7-dimensional Euclidean space. We found that while some proteins moved between organelles, others remained classified within the same organelle but moved as a group relative to other organelles. The expression of HIV-1 affected the distribution of peroxisomal proteins, causing them to move closer in analytic space to proteins of the endoplasmic reticulum and lysosomes, but only when the viral protein Nef was expressed. These data identify Nef-dependent changes in peroxisomes as a novel perturbation of the host cell by HIV-1.