This study examined the genetic variation within the gene trefoil factor 3 (TFF3) in relation to unexplained female infertility in a group of women where aberrant endometrial maturation was suspected. The study consisted of 113 women with a diagnosis of unexplained infertility and 289 healthy fertile volunteers. Five single nucleotide polymorphisms rs225439, rs533093, rs225361, rs11701143, and rs77436142 within TFF3 gene were analyzed using real-time PCR. The formed haplotype pattern within the TFF3 gene in relation to infertility was also assessed. TFF3 protein localization and expression in receptive stage endometrium at the time of implantation was measured in a subset of fertile (n = 7) and infertile (n = 12) women. Allele and genotype frequencies did not differ significantly between fertile and infertile women, nor did the formed haplotypes. TFF3 protein was expressed in all cell types in receptive stage endometria in fertile and infertile women. No significant association was observed between protein expression and analyzed genotypes. A significantly higher TFF3 expression in luminal epithelial cells was detected in women with unexplained infertility (p = 0.003).