The study purpose was to evaluate the reproductive experience, specifically cycle characteristics and treatment outcomes, of lesbian women. In addition, we aimed to determine whether there are differences in pregnancy outcomes when comparing lesbian women undergoing ovulation induction (OI) versus natural cycles with donor intrauterine insemination (IUI), as well as lesbian and heterosexual women undergoing the same assisted reproductive technology treatment. This was a retrospective cohort study including women who underwent an IUI with cryopreserved sperm between 2006 and 2018. The primary outcome of interest was clinical pregnancy (CP) rate. A total of 216 lesbian women (451 natural cycles and 441 OI cycles) and 584 heterosexual women (1177 natural cycles and 1238 OI cycles) were included in the study. Thirty percent of lesbian women had a hysterosalpingogram as part of their initial workup. Approximately 40% of lesbian women who underwent OI/IUI had previously undergone at least one natural cycle/IUI. There was no significant difference in CP rate when comparing lesbian women and heterosexual women undergoing natural or OI/IUI, or when comparing lesbian women who underwent natural versus OI/IUI cycles. However, there was a significantly higher multiple gestation rate among lesbian women undergoing OI compared with those undergoing natural cycles (11.8% vs. 0%, p = 0.01). This large study showed that while pregnancy outcomes were similar between groups, the multiple gestation rate was higher in lesbian women undergoing OI compared with lesbian women undergoing natural cycles.