Spermatogonial stem cells (SSCs) self-renew and contribute genetic information to the next generation. Inducing directional differentiation of porcine SSCs may be an important strategy in exploring the mechanisms of spermatogenesis and developing better treatment methods for male sterility. Here, we established an in vitro culture model for porcine small seminiferous tubule segments, to induce SSCs to differentiate into single-tail haploid spermatozoa. The culture model subsequently enabled spermatozoa to express the sperm-specific protein acrosin, and oocytes to develop to blastocyst stage after round spermatid injection. The addition of retinoic acid (RA) to the differentiation media promoted the efficiency of haploid differentiation. RT-PCR analysis indicated that RA stimulated the expression of Stra8 but reduced the expression of NANOS2 in spermatogonia. Genes involved in post-meiotic development, Prm1 and Tnp1, were up-regulated in the presence of RA. The addition of RAR inhibitor, BMS439, showed that RA enhanced the expression of cAMP responsive-element binding protein through RAR, and promoted the formation of round spermatids.