Equine semen is commonly frozen in 0.5-mL straws at a concentration of 400 million spermatozoa per mL. Equine pregnancies have been produced in the past few years using assisted reproduction techniques that require very few sperm, such as sperm injection and gamete intrafallopian transfer. Consequently, thawing even a single straw would waste a high percentage of the sperm. The goals of this study were to: (1) evaluate the motility and viability of cryopreserved spermatozoa that had been thawed and refrozen, and (2) compare the effect of serial dilution on refreezing parameters. It was hypothesized that both motility and viability would be significantly decreased as a consequence of refreezing, but sufficient live, motile spermatozoa would be present for assisted reproductive techniques. Semen from six stallions of mixed light horse breeds was collected, evaluated, and frozen in lactose-EDTA in 0.5-mL straws. Semen from each stallion was subsequently thawed, held at room temperature for 10 minutes, and then refrozen in 0.25-mL straws. Additional semen from the initial freeze was thawed, diluted in the original extender, and refrozen at concentrations of 40 × 10⁶, 4 × 10⁶, 4 ×  10⁵, and 4 × 10⁴ spermatozoa per mL. Refrozen semen was evaluated for motility visually and by CASA. Viability of refrozen semen was determined by flow cytometry following staining with propidium iodide (PI). Percentages of motile spermatozoa prior to freezing, after the first freezing, and after the second freezing were compared using the PROC GLM model in SAS. All values are presented as the mean ± standard deviation. Total motility decreased sigificantly (P < 0.05) from 91.8 ± 3.1% prior to freezing to 64.2 ± 7.7% after the first freezing and 45.7 ± 10.4% after the second freezing. However, only a tendency (P = 0.09) toward a decrease in sperm viability following refreezing as measured by PI staining was noted. Dilution of sperm prior to refreezing did not significantly effect (P > 0.05) subsequent motility or viability parameters. This study demonstrated that refrozen equine semen retained approximately 70% of the initial post-thaw motility. Dilution prior to refreezing would allow for the reallocation of a single 0.5-mL straw into hundreds or thousands of smaller straws for future use in assisted reproduction. Refreezing would allow for the judicious use of valuable stallion semen in limited supply.