Lysine specific demethylase 5B gene (KDM5B, also known as JARID1B or PLU-1), encoding an enzyme of the lysine-specific histone demethylase family, has been reported to regulate androgen receptor transcriptional activity and male reproduction. To fully study the expression characteristics and genetic effects of pig KDM5B gene, the objective of this study was to investigate the mRNA expression profiles of KDM5B among different tissues and testicular cells (spermatogonia stem cells, SSCs; sertoli cells, SCs; leydig cells, LCs), as well as to explore the insertion/deletion (indel) variations of this gene. Expression analysis results revealed that the KDM5B gene was highly expressed in testis than other tissues in 7-day-old piglet (P < 0.01). In particular, this gene was highest expressed in testis of adult boar (P < 0.01). Furthermore, the KDM5B mRNA expression level in SSCs was significantly higher than those in LCs and SCs (P < 0.01). Besides, a 35-bp indel was first verified in the intron 11 of pig KDM5B gene, and the animals with insertion/insertion genotype exhibited superior testicular weight and testicular short perimeter than those with other genotypes (P < 0.05) in 40-day-old Landrace pigs. Together, these findings suggest that KDM5B plays a crucial role in male fertility and the 35-bp indel can be used for marker-assisted selection of boar. •The KDM5B gene was highly expressed in testis of male piglets and adult pigs (P < 0.01).•The pig KDM5B gene was highly expressed in spermatogonia stem cells (P < 0.01).•The 35-bp indel was firstly verified to associate with pig testicular morphology traits (P < 0.05).