The matrix metalloproteinase 23 (MMP-23), which might play a role in ovulation in mammals, was one of the promising candidate genes for litter size traits in pigs. In the present research, partial sequence of porcine MMP-23 (pMMP-23) gene, including exons 2-8 (GenBank: EU360790), was obtained. Real-time PCR analysis revealed that pMMP-23 gene was highly expressed in ovary. PCR-Sau3A I-RFLP and PCR-Acc II-RFLP assay were established to detect a C/T mutation in exon 3 (EU360790: g. 269C>T) and an A/G mutation in exon 4 (EU360790: g. 505A>G), respectively. Association study for these two SNPs with litter size was assessed in three independent populations (Minzhu, Landrace I and Landrace II). Statistical analysis demonstrated that for second and subsequent litters, TT sows produced more TNB than CC pigs in Landrace breed (P<0.05) at g. 269C>T locus, and the additive effect was significant (P<0.05); GG sows produced more TNB and NBA than AA pigs in Minzhu (P<0.01) and Landrace breeds (P<0.05) at g. 505A>G locus, and the additive effect was significant (P<0.01 or P<0.05). Our study suggested that the pMMP-23 gene might be a novel candidate gene for litter size traits, and g. 505A>G locus might be a useful molecular marker for marker assisted selection (MAS).