Prostate cancer (PCa) is the second leading cause of cancer-related deaths in men. Genome-wide association studies have identified two non-synonymous single nucleotide polymorphisms (SNPs), rs61752561:G>A (D84N) and rs17632542:T>C (I161T), within the prostate-specific antigen (PSA)/Kallikrein-3 (KLK3), the current diagnostic marker for PCa. PSA has a role in PCa onset and metastasis. The two SNPs are associated with lower serum-PSA levels and reduced PCa risk. It is unclear if their association with PCa risk is due to lower serum-PSA levels in individuals with susceptibility alleles or have a biological role in prostate pathogenesis. Cell-based studies suggested that the two SNPs reduce the proliferative and migratory capabilities of PC3 cells and in HUVEC cell angiogenesis models, compared to the wild-type PSA expressing cells which may underpin the reduced risk in patients harbouring the susceptibility alleles. Biochemical-assays suggested that the SNP isoforms have a lower proteolytic activity and the T161-PSA variant has a lower protein stability that may also correlate to low serum-PSA levels while the N84-PSA leads to an extra-glycosylation site. Gene expression data from tumour tissues, verified the lower expression of KLK3 mRNA and a KLK3 splice variant for C-allele of the rs17632542 SNP. In conclusion, we have comprehensively investigated the molecular consequences of the two PSA germline variants which may contribute to a potential role in PCa pathogenesis. Further, utilisation of this information into current PSA diagnostic practice may improve the predictive accuracy of the test.