MicroRNAs (miRNAs) are 20-25 nt, endogenous non-coding RNA molecules that act by binding to the complementary sequence of target messenger RNAs. Many evidences showed that miRNAs were involved in the process of germ proliferation and differentiation. In the present study, miR-27a gene was selected as a candidate gene for litter size due to its biological function, its location near a mummified pigs QTL, and its differentially expressed profile in Large White and Chinese Erhualian PMSG-hCG stimulated preovulatory ovaries. By comparative sequencing of miR-27a gene in Large White and Chinese Meishan pigs, one SNP (T/C) which created an additional HpaII site was detected. Then associations of this SNP with litter size traits were assessed in Large White (n = 142) and DIV (n = 140) pig populations. The statistical analysis demonstrated that AA differed from AB (P < 0.01) and BB (P < 0.05) for total number of piglets born in the first parities, and also differed from AB (P < 0.01) for the number of piglets born alive in all parities (P < 0.05) in DIV pigs. No significant difference was observed between different genotypes in Large White pigs.