This study aimed to correlate the influence of PON1 activity with Computer Assisted Semen Analysis (CASA) system evaluation parameters and sperm viability of in natura porcine semen and to investigate PON1 expression (mRNA) in testis and epididymis tissue of adult porcine sire. The sperm fraction (SF) showed the highest PON1 activity and males 81, 80 and 79 (P<0.05) showed the highest PON1 activity. When the ejaculate was diluted (1:1) it showed an average reduction of 44% in PON1 activity. PON1 activity in FE showed a positive correlation with sperm concentration, DCL, VCL and IDNA (P<0.05) and showed a negative correlation for STR and LIN parameters assessed by CASA. The mRNA expression was only observed for the body portion of the epididymis. The FE is the part of the ejaculate that most showed PON1 activity and the parameters DCL, VCL, STR and LIN, together with sperm concentration and viability (DNA integrity) are the ones that showed the highest association with the enzyme. On the other hand, PON1 was only expressed (mRNA) in the epididymal body of the porcine sire.