Dio3os is a significant lncRNA gene located in the Dlk1-Dio3 imprinted locus, and plays an important role in the body growth and development. However, there is relatively little information regarding the effects of this gene on reproductive activity in mice. Thus, the aim of this study was to investigate the effects of Dio3os on growth, development and reproductive activity in mice. Moreover, expression levels of associated genes were also monitored, using mouse endometrial stromal cells (ESCs). Firstly, qPCR and Western Blot were used to determine the expression levels of genes associated with Dio3os, using mouse ESCs. Then, RNA-seq was utilized to classify and annotate the activity of differentially expressed genes affected by Dio3os. Moreover, RNA pull-down and mass spectrometry were used to identify and analyze Dio3os-binding proteins. Additionally, 24 female mice were randomly divided into 4 equal groups (n=6 each), including LV3-Dio3os, LV3-NC, LV5-Dio3os and LV5-NC. Data of body weight, body length, tail length, occurrence rate of estrous cycle and organ coefficients for these mice were recorded. Results of qPCR and Western Blot showed that Dio3os affected the expression levels of Dlk1, Gtl2, Dio3 and genes related to development and apoptosis. Besides, Dio3os was highly expressed in mouse uterus and ovary. This gene also increased body weight and body length and affected the organ coefficients. Moreover, Dio3os increased the occurrence rate of estrus and correspondingly decreased the occurrence rate of diestrus. Together, these findings indicate that Dio3os affects growth, development and reproductive activity in mice.