APOL1 harbors C-terminal sequence variants (G1 and G2), which account for much of the increased risk for kidney disease in sub-Saharan African ancestry populations. Expression of the risk variants has also been shown to cause injury to podocytes and other cell types, but the underlying mechanisms are not understood. We used Drosophila melanogaster and Saccharomyces cerevisiae to help clarify these mechanisms. Ubiquitous expression of the human APOL1 G1 and G2 disease risk alleles caused near-complete lethality in D. melanogaster, with no effect of the G0 nonrisk APOL1 allele, corresponding to the pattern of human disease risk. We also observed a congruent pattern of cellular damage with tissue-specific expression of APOL1. In particular, expression of APOL1 risk variants in D. melanogaster nephrocytes caused cell-autonomous accumulation of the endocytic tracer atrial natriuretic factor-red fluorescent protein at early stages and nephrocyte loss at later stages. We also observed differential toxicity of the APOL1 risk variants compared with the APOL1 nonrisk variants in S. cerevisiae, including impairment of vacuole acidification. Yeast strains defective in endosomal trafficking or organelle acidification but not those defective in autophagy displayed augmented APOL1 toxicity with all isoforms. This pattern of differential injury by the APOL1 risk alleles compared with the nonrisk alleles across evolutionarily divergent species is consistent with an impairment of conserved core intracellular endosomal trafficking processes. This finding should facilitate the identification of cell injury pathways and corresponding therapeutic targets of interest in these amenable experimental platforms.