The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Here, we demonstrate that human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells.