Peroxisomes are single-membraned organelles ubiquitous to eukaryotic cells that house metabolic reactions that generate and destroy harmful oxidative intermediates. They are dynamic structures whose morphology, abundance, composition, and function depend on the cell type and environment. Perhaps due to the potentially damaging and protective metabolic roles of peroxisomes and their dynamic presence in the cell, peroxisome biogenesis is emerging as a process that involves complex underlying mechanisms of regulated formation and maintenance. There are roughly 30 known peroxins, proteins involved in peroxisome biogenesis, many of which have been conserved from yeast to mammals. This review focuses on the biogenesis of peroxisomes with an emphasis on the regulation of peroxisome formation and the import of peroxisomal matrix proteins in the model organism Saccharomyces cerevisiae.