Mesenchymal stromal cell (MSC) are multipotent precursor cells that have been isolated from many tissues, including endometrium in some species. These cells are necessary for tissue homeostasis, which in the cycling equine endometrium is regulated in part by changes in concentration of steroid hormones. The expression of oestrogen and progesterone receptors during the oestrous cycle has been studied before, but MSC gene expression is not reported as well as the effects of steroid hormones on in vitro proliferation of endometrial MSC. This study was designed to investigate the influence of steroid hormones on endometrial MSC proliferation in vitro and to examine mRNA expression of MSC markers (CD29, CD44, CD73, CD90, and CD105) in the healthy equine endometrium during the oestrous cycle. Equine endometrial tissue was collected postmortem (n = 6) and digested using a dissociation medium and mucin-1-bound magnetic beads were utilised to remove epithelial cells from the resulting single-cell solution. The cells were expanded in culture and, at passage 4, incubated with 3 different concentrations of oestradiol and progesterone for 5 days. For the proliferation analysis the Alamar Blue® assay was used according to manufacturer instructions. Endometrial biopsies, for quantitative RT-PCR analysis, were taken from healthy mares (n = 5) on Day 5 and 13 post-ovulation, during oestrus (1 follicle >3.5cm, pronounced uterine oedema), and seasonal anestrous (seasonal anovulation). The ΔCt values were used for statistical analysis using SPSS Statistics 22 (IBM Corp., Armonk, NY). Data for quantitative PCR are presented as gene expression relative to the mean of 18S and GAPDH. No significant differences in proliferation could be detected in the various groups incubated with steroid hormones compared with the controls supplemented with charcoal-stripped fetal bovine serum. Detectable levels of mRNA for all 5 MSC markers analysed were present throughout the oestrous cycle. While the levels of CD73 were consistent, the expression of 3 MSC markers (CD29, CD44, and CD105) was elevated at Day 13. This difference was substantial between Day 13 and oestrus for CD29 (37.6 ± 6.2 and 12.2 ± 3.4; P < 0.01) and CD105 (8.3 ± 0.9 and 4.5 ± 0.6; P < 0.05), and between Day 5 and 13 for CD29 (7.4 ± 2.3 and 37.6 ± 6.2) and CD44 (12.9 ± 1.8 and 4.1 ± 0.3; P < 0.01). In contrast, CD90 expression was higher at oestrus (27.8 ± 3.8) than at Day 5 (6.7 ± 0.9) or 13 (12.0 ± 2.1; P < 0.01). Elevated quantities of MSC marker transcripts during late diestrus might be linked to the preparation of the equine endometrium for the proliferation phase associated with oestrus. However, the in vitro proliferation of endometrial-derived MSC is not influenced by the steroid hormones, although gene expression of steroid hormone receptors is present throughout the oestrous cycle of the mare. In summary, this study shows that the equine endometrium expresses MSC markers, and it does so at variable levels throughout the oestrous cycle; however, cell proliferation in vitro is not influenced by steroid hormones. This information will be useful for future studies aiming to derive endometrial MSC from mares.