Peroxisomes are core eukaryotic organelles that generally function in lipid metabolism and detoxification of reactive oxygen species, but they are increasingly associated with taxa-specific metabolic, cellular, and developmental functions. Here, we present a brief overview of peroxisome assembly, followed by a discussion of their functional diversification. Matrix protein import occurs through a remarkable translocon that can accommodate folded and even oligomeric proteins. Metabolically specialized peroxisomes include glycosomes of trypanosomes, which have come to compartmentalize most of the glycolytic pathway and play a role in developmental signal transduction. The differentiation of physically distinct subcompartments also contributes to peroxisome diversification; in the clade of filamentous ascomycetes, dense-core Woronin bodies bud from peroxisomes to gate cell-to-cell channels. Here, the import of oligomeric cargo is central to the mechanism of subcompartment specification. In general, the acquisition of a tripeptide peroxisome targeting signal by nonperoxisomal proteins appears to be a recurrent step in the evolution of peroxisome diversity.