Prostaglandin D and the associated prostaglandin D synthases (PGDS) and receptor (DP) are considered to be involved in spermatogenesis. However, the interplay of the PGDS-DP system in male reproduction is far from being understood. The expression of PGDS lipocalin (L) and hematopoietic (H) type and DP was studied in the GnRH agonist–downregulated canine testis (week, w 0) and during recrudescence of spermatogenesis after implant removal (w 3, 6, 9, 12). H-PGDS, L-PGDS and DP were present in the adult (CG), juvenile (JG) and downregulated canine testis at the mRNA level. PGDS immunohistochemistry revealed positive staining in the cytoplasm of Leydig cells (LCs) of all samples i.e., no difference between groups. mRNA expression (ratio) of L-, H-PGDS and DP did not differ between groups w 0–12 and CG. In contrast, significant differences were found for L-PGDS (p = 0.0388), H-PGDS (p < 0.001) and DP (p < 0.001) for the groups at downregulation (w0, suprelorin group, SG, profact group, PRG) compared with the control groups (JG, CG). L-PGDS expression was lowest in JG, whereas H-PGDS was significantly lower in CG compared with JG and at downregulation (p < 0.001 to p < 0.01). The highest ratio for H-PGDS and DP was observed in the dogs treated with buserelin acetate (PRG). Our data show that the PGDS-DP system is expressed in juvenile and adult canine testes and that downregulation of the testicular endocrine and germinative function significantly affects H-PGDS, L-PGDS and DP mRNA expression indicating a role in the regulation of spermatogenesis.