Domain shuffling has provided extraordinarily diverse functions to proteins. Nevertheless, how newly combined domains are coordinated to create novel functions remains a fundamental question of genetic and phenotypic evolution. Previously, we reported a unique mechanism of gene creation, whereby new combinations of functional domains are assembled from distinct genes at the RNA level, reverse transcribed, and integrated into the genome by the L1 retrotransposon. The novel gene PIPSL, created by the fusion of phosphatidylinositol-4-phosphate 5-kinase (PIP5K1A) and 26S proteasome subunit (S5a/PSMD4) genes, is specifically transcribed in human and chimpanzee testes. We present the first evidence for the translation of PIPSL in humans. The human PIPSL locus showed a low nucleotide diversity within 11 populations (125 individuals) compared with other genomic regions such as introns and overall chromosomes. It was equivalent to the average for coding sequences or exons from other genes, suggesting that human PIPSL has some function and is conserved among modern populations. Two linked amino acid-altering single-nucleotide polymorphisms were found in the PIPSL kinase domain of non-African populations. They are positioned in the vicinity of the substrate-binding cavity of the parental PIP5K1A protein and change the charge of both residues. The relatively rapid expansion of this haplotype might indicate a selective advantage for it in modern humans. We determined the evolutionary fate of PIPSL domains created by domain shuffling. During hominoid diversification, the S5a-derived domain was retained in all lineages, whereas the ubiquitin-interacting motif (UIM) 1 in the domain experienced critical amino acid replacements at an early stage, being conserved under subsequent high levels of nonsynonymous substitutions to UIM2 and other domains, suggesting that adaptive evolution diversified these functional compartments. Conversely, the PIP5K1A-derived domain is degenerated in gibbons and gorillas. These observations provide a possible scheme of domain shuffling in which the combined parental domains are not tightly linked in the novel chimeric protein, allowing for changes in their functional roles, leading to their fine-tuning. Selective pressure toward a novel function initially acted on one domain, whereas the other experienced a nearly neutral state. Over time, the latter also gained a new function or was degenerated.