Long noncoding RNAs (IncRNAs) have been identified in all eukaryotes and are most abundant in the human genome. However, the functional importance and mechanisms of action for human IncRNAs are largely unknown. Using comparative sequence, structural, and functional analyses, we characterize the evolution and molecular function of human IncRNA JPX. We find that human JPX and its mouse homolog, IncRNA Jpx, have deep divergence in their nucleotide sequences and RNA secondary structures. Despite such differences, both IncRNAs demonstrate robust binding to CTCF, a protein that is central to Jpx's role in X chromosome inactivation. In addition, our functional rescue experiment using Jpx-deletion mutant cells shows that human JPX can functionally complement the loss of Jpx in mouse embryonic stem cells. Our findings support a model for functional conservation of IncRNAs independent from sequence and structural divergence. This study provides mechanistic insight into the evolution of IncRNA function. (C) 2019 Elsevier Ltd. All rights reserved.