Abstract. Spermatogenesis-associated, serine-rich 2-like (SPATS2L) is an important reproduction-related gene. In this study, the full-length cDNA sequence of the porcine SPATS2L gene was cloned through the RACE (rapid amplification of cDNA ends) method. The porcine SPATS2L gene encodes a protein of 559 amino acids which shares high homology with the SPATS2L proteins of seven species: dog (94 %), white-tufted-ear marmoset (91 %), human (92 %), rhesus monkey (92 %), horse (92 %), rat (88 %) and mouse (88 %). This novel porcine gene was assigned to GeneID: 100415809. The phylogenetic analysis revealed that the porcine SPATS2L gene has a close genetic relationship with the canine SPATS2L gene. PCR-Pst I-RFLP was established to detect GU474997:c.1687 C > T substitution of porcine SPATS2L gene mRNA, and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this single-nucleotide polymorphism (SNP) with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows (P < 0.01). Therefore, SPATS2L gene could be an useful candidate gene in selection for increasing litter size in pigs.