Premature ovarian insufficiency (POI) is a major cause of female subfertility. Although POI affects approximately 1-2% women worldwide, the etiology of a large number of POI patients remains unknown partially due to the genetic heterogeneity of POI. EIF4ENIF1 is one of the known POI-causative genes, and it plays an essential role in inhibiting mRNA translation and regulating mRNA destabilization in ovarian cells. In our study, two EIF4ENIF1 variants, c.9_11delGAG (p.R4del) (rs3834682) and c.2861G > C (p.G954A) (rs766008983) were identified in two sporadic Han Chinese POI patients through whole-exome sequencing. Both variants are rare in the human population. The two patients' mothers don't carry the rare variants and they have regular menstruation. The missense variant c.2861G > C was predicted to be deleterious by multiple bioinformatic tools. Western blot analysis further demonstrated that both of the two variants exhibited reduced mRNA and protein expression levels compared with the wild-type in vitro. Taken together, our findings reported two rare POI-associated EIF4ENIF1 variants, providing insights into genetic counseling and suggesting the contribution of EIF4ENIF1 variants in female infertility.