The antioxidant enzyme paraoxonase-1 (PON1) may limit oxidative stress in the development of cardiovascular diseases (CVD) and obstructive sleep apnea (OSA). The aim of the study was to determine PON1 gene L55M polymorphism in OSA-positive and OSA-negative subjects, along with paraoxonase activity of the enzyme (PON1-act). Caucasians aged 25-75, with BMI 19.0-53.0 kg/m2 and no acute or severe chronic disorder underwent polysomnography, and OSA-negative (n = 44) and OSA-positive (n = 57) groups were established. The following parameters were assessed: arterial blood pressure and serum glucose, lipids, C-reactive protein, and homocysteine. Genomic DNA was extracted and amplified, and automatic sequencing was used to confirm the LL, LM, MM genotypes. PON1-act was measured spectrophotometrically using paraoxon as a substrate. We found that frequency of polymorphisms differed significantly between the OSA-negative and OSA-positive patients (p < 0.05). Increased PON1-act was observed in the LL-genotype versus the LM + MM-genotype in the study population (p < 0.05). PON1-act was higher in the OSA-negative compared with OSA-positive patients (p < 0.001); in general and in the subgroups presenting the LL or LM genotype. In addition, there was an inverse relationship between PON1-act and LDL-cholesterol in the entire study population. The OSA-positive group presented an inverse relationship between PON1-act and fasting glucose. We conclude that patients could benefit from the LL genotype related with higher activity of PON1. OSA pathology might decrease the enzyme activity, despite the presence of L allele.