Decidualization is a critical process for successful embryo implantation and subsequent placenta formation. The characterization and physiological function of long non-coding RNA (lncRNA) during decidualization remain largely unknown. In the present study, we conducted RNA-sequencing analysis to compare gene expression between decidua of days six and eight, and normal pregnant endometrium (day four). A total of 2,332 high-confidence putative lncRNA transcripts were expressed. Functional clustering analysis of cis and trans lncRNA targets showed that differentially expressed lncRNAs may regulate multiple gene ontology terms and pathways that have important functions in decidualization. Subsequent analyses using qRT-PCR validated that eight of all lncRNAs were differentially regulated in mice uteri during decidualization, both in vivo and in vitro. Furthermore, we showed that differentially expressed lncRNA of Hand2os1 was specifically detected in stromal cells on days two to five of pregnancy and was strongly up-regulated in decidual cells on days six to eight of pregnancy. Similarly, Hand2os1 expression was also strongly expressed in decidualized cells following artificial decidualization, both in vivo and in vitro. In uterine stromal cells, P4 was able to significantly up-regulate the expression of Hand2os1, but up-regulation was impeded by RU486, whereas E2 appeared to have no regulating effect on Hand2os1 expression. Concurrently, Hand2os1 significantly promoted the decidual process in vitro and dramatically increased decidualization markers Prl8a2 and Prl3c1. Our results provide a valuable catalog for better understanding of the functional roles of lncRNAs in pregnant mouse uteri, as it relates to decidualization.