Recent progress in DNA sequencing technology has made whole genome sequences available from a wide variety of vertebrates. Genome-wide comparisons have revealed characteristics of the evolutionary change of different aspects of genomes involving chromosomes, genes, regulatory regions, and retrotransposons. Various mutations have occurred during evolution among orthologues, genes in different species that diverged from a common ancestral gene by speciation. A major difference between mammals and other vertebrates is the presence or absence of homopolymeric amino acid repeats (sequences without interruptions in the run of a single amino acid residue), indicating that these repeats were acquired specifically in the mammalian lineage. Transcription factor Brn-2/Pou3f2 is prominently expressed in the neocortex. The neocortex has undergone pronounced expansion and development during evolution, and is considered to be responsible for cognitive function, sensory perception, and consciousness. Mammalian Brn-2 has glycine, glutamine, and proline repeats, whereas most or all of these repeats are absent from Brn-2 orthologues in non-mammalian vertebrates. Here we report the remarkable deterioration of a characteristic mammalian maternal behavior, pup retrieval, in non-mammalized mice, in which the transcription factor Brn-2 was replaced with the Xenopus tropicalis orthologue lacking all of the homopolymeric amino acid repeats of mammalian Brn-2. These mice with a non-mammalized Brn-2 gene show less curiosity towards their pups and drastically decreased pup retrieval behavior that leads to increased pup death. Quantitative immunohistochemical analysis revealed decreases in the rate-limiting enzymes of dopamine and serotonin synthesis in various brain structures. This study indicates that pup retrieval behavior in mammals is thus strongly related to monoamine neurotransmitter levels via the acquisition of homopolymeric amino acid repeats during mammalian evolution.