Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) and infection frequently results in reproductive tract (RT) sequelae such as pelvic inflammatory disease and infertility. However, the immune factors and processes involved in the clearance and immunopathology of Chlamydia infection are not well understood. In previous studies we showed that interferon (IFN)-ε, a novel type I IFN that is exclusively and constitutively expressed in the female RT, plays an important role in protecting against Chlamydia infections at the earliest stages of infection. Here, we examined the effects of IFN-ɛ on innate cellular responses in the female RT in order to elucidate the potential mechanisms that underpin how IFN-ɛ protects against Chlamydia infections. Female wild type and IFN-ε deficient (-/-) C57BL/6 mice were pre-treated with progesterone to synchronise their oestrous cycles and prime for infection. Seven days later, mice were infected intra-vaginally with Chlamydia muridarum or sham-infected. Uterine horns were harvested at 3 days post infection and the effects of IFN-ε deficiency on Chlamydia infection, immune factor expression and cellular infiltration were assessed using real-time qPCR and flow cytometry. We show that IFN-ε-/- mice have increased Chlamydia 16S expression in the upper RT. This corresponded with fewer NK cells, which are known to play a role in protecting against Chlamydia through the production of IFN-γ. IFN-γ producing CD45+ cells were decreased in the infected IFN-ε-/- mice, of which over 60% were NK cells. Tissue-resident uterine (u)NKs were also decreased in these mice. The changes in IFN-γ+ and NK cells were associated with reduced iNOS expression. These findings suggest that IFN-ε may protect against Chlamydia RT infections by potentiating the recruitment of protective IFN-γ-producing NK cells and increasing iNOS.