Mutations in the gene encoding bilirubin UDP-glucuronosyltransferase (UGT1A1) are known to cause Crigler-Najjar syndrome type II (CN-II). We previously encountered a patient with a nonsense mutation (Q331X) on one allele and with no other mutations in the promoter region or other exons, and proposed that CN-II is inherited as a dominant trait due to the formation of a heterologous subunit structure comprised of the altered UGT1A1 gene product (UGT1A1-p.Q331X) and the intact UGT1A1. Here, we investigated the molecular basis of CN-II in this case by expressing UGT1A1-p.Q331X in cells. UGT1A1-p.Q331X overexpressed in Escherichia coli or mammalian cells directly bound or associated with intact UGT1A1 in vitro or in vivo, respectively. Intact UGT1A1 was observed as a dimer using atomic force microscopy. Fluorescent-tagged UGT1A1-p.Q331X and intact UGT1A1 were colocalized in 293T cells, and fluorescence recovery after photobleaching analysis showed that UGT1A1-p.Q331X was retained in the endoplasmic reticulum (ER) without rapid degradation. These findings support the idea that UGT1A1-p.Q331X and UGT1A1 form a dimer and provide an increased mechanistic understanding of CN-II.