Matrix metalloproteinase (MMP)-2 and -9 are gelatinases which are capable of degrading type IV collagen and have been linked to cancer invasion and metastatic development. MMP-2 and MMP-9 gene polymorphisms may affect their biological function, and thus their role in cancer development and progression. We analyzed the association of the polymorphism frequencies ofMMP-2-735C/T andMMP-9-1562C/T with MMP-2 and MMP-9 serum concentrations, as well as their potential effects in lung cancer patients. We conducted a retrospective, case-control study consisting of 112 lung cancer patients and 100 healthy individuals from a Caucasian population in Poland. Polymerase chain reaction with restriction fragment length polymorphism (PCR/RFLP) and electrophoresis was used to genotype genomic DNA from whole blood samples. MMP-2 and MMP-9 serum concentrations were then determined using ELISA. For statistical analysis, Statistica version 13 from TIBCO Software Inc. was utilized with a significance level <0.05. Logistic regression analysis revealed thatMMP-2-735CC (OR = 5.39; 95% CI = 0.62–47.17;p= 0.238504) and -735CT genotype (OR = 7.22; 95% CI = 0.78–67.14;p= 0.072836), as well asMMP-9-1562CC (OR = 1.45; 95% CI = 0.31–6.70;p= 0.757914) and -1562CT genotype (OR = 1.60; 95% CI = 0.33–7.83;p= 0.548801) were associated with a higher risk of lung cancer. There were statistically significant differences observed in the MMP-2 concentration between individuals with the -735CC genotype and the -735CT genotype (non-smoking control: 204.04 ng/mL vs. 237.00 ng/mL, respectively,p= 0.041479; adenocarcinoma patients: 157.69 ng/mL vs. 126.37 ng/mL, respectively,p= 0.013222), as well as differences in the MMP-9 concentration between individuals with the -1562CC genotype and the -1562CT genotype (smoking control: 385.67 ng/mL vs. 562.80 ng/mL, respectively,p= 0.000936; patients with other lung neoplasms: 821.64 ng/mL vs. 928.88 ng/mL, respectivelyp= 0.023315). The role ofMMP-2-735C/T andMMP-9-1562C/T polymorphisms in an increased risk of lung cancer cannot be dismissed. Specific genotypes affect MMP-2 and MMP-9 concentrations in both lung cancer patients and healthy controls, which may thereby increase lung cancer risk, disease aggressiveness, and patient survival outcomes.