Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten’s). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively, t-test p < 0.003, n = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.