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Steroid hormone levels are associated with estrous behavior, which affects timely mating and reproductive efficiency in pigs. 17β-hydroxysteroid dehydrogenase type 14 (<i>HSD17B14</i>) modulates steroid synthesis and metabolism. To identify the functional single nucleotide polymorphisms (SNPs) in the porcine <i>HSD17B14</i> gene, ear tissues from Large White and Mi gilts were collected to extract genomic DNA. Variable lengths of truncated promoter of <i>HSD17B14</i> gene were used to determine the promoter activity by a dual luciferase reporter system. The vector <i>HSD17B14</i>^Phe or <i>HSD17B14</i>^Val was transfected into porcine granulosa cells (GCs). The core promoter region was identified between −72 bp and −218 bp. Six of seven SNPs had significant differences of allele frequency between Large White and Mi gilts. The plasmids with the wild genotype AA of rs329427898 maintained a smaller fraction of promoter activity compared with the plasmids with the mutant genotype GG, while the plasmids with wild the genotype TT of rs319864566 had a greater promoter activity than the plasmids with the mutant genotype CC. A missense mutation (Phe73Val) caused changes in the structural dynamics and function of the <i>HSD17B14</i> protein. The highly expressed <i>HSD17B14</i>^Val degraded less estradiol into estrone, while the relatively lowly expressed <i>HSD17B14</i>^Phe degraded more estradiol into estrone, suggesting the protein activity of <i>HSD17B14</i>^Phe was greater than that of <i>HSD17B14</i>^Val. Moreover, the <i>HSD17B14</i>^Phe group has a greater apoptosis rate of porcine GCs. The <i>HSD17B14</i> gene could been used as a candidate molecular marker for estrus behavior in pigs.