SP110 has previously shown to be a genetic determinant of host resistance to the intracellular pathogen infection in mouse and human. However, its relevant biological information in large non-primate animals still remains unknown. Here we report the novel discovery and characterization of three transcript variants of horse SP110. The transcript variant 1 (Tv1) of horse SP110 with the longest open reading frame has four domains (Sp100, SAND, PHD and Bromo domain). Tv2 and Tv3 share the same N-terminal sequence as Tv1, which contains Sp100 and SAND. We show that Tv2 is generated from alternative splicing and deletion of Exon17-Exon18 segment, while Tv3 is generated by pre-mature transcriptional termination at Exon 16. Furthermore, we demonstrate that the heterologous expression of horse SP110 variants stimulate macrophages into an activation-like phenotype. The macrophages underwent a shift in enhancing the secretion of cytokines (interleukin-1 (IL-1) and TNF-α) and accelerating inducible nitric oxide synthase (iNOS) activity, and eventually went into apoptotic cell death. Intriguingly, horse SP110 Tv1 showed more capability to trigger the immune activities compared to Tv2 and Tv3. To our knowledge, the identification of SP110 transcript variants from horse is the first report on biological function of SP110 in perissodactyla animals.