BackgroundTransvaginal ultrasound‐guided aspiration (TUA) is used for post‐fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA.ObjectivesTo evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA.Study designRetrospective case series.MethodsData were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5–7 days and 3–4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow‐up communication. The effects of mare, gestation‐ and TUA‐related variables on pregnancy and foaling rates were analysed by the chi‐square‐test for homogeneity and Fisher's exact test and logistic regression.ResultsTUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5–7 days after TUA was performed. At 3–4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25–35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares.Main limitationsMissing retrospective data despite extensive follow‐up.ConclusionThis is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).