Human genome projects have enabled whole genome mapping and improved our understanding of the genes in humans. However, many unknown genes remain to be functionally characterized. In this study, we characterized human chromosome 4 open reading frame 34 gene (hC4orf34). hC4orf34 was highly conserved from invertebrate to mammalian cells and ubiquitously expressed in the organs of mice, including the heart and brain. Interestingly, hC4orf34 is a novel ER-resident, type I transmembrane protein. Mutant analysis showed that the transmembrane domain (TMD) of hC4orf34 was involved in ER retention. Overall, our results indicate that hC4orf34 is an ER-resident type I transmembrane protein, and might play a role in ER functions including Ca2+ homeostasis and ER stress.