Atherosclerosis is a multifactorial disease influenced by genetic and lifestyle factors (e.g., smoking). The rs1799963 G/A polymorphism in the prothrombin (F2) gene is associated with thrombosis and cardiovascular diseases. However, the interaction between this genetic variant and smoking on the risk of atherosclerosis has not been thoroughly investigated. This study aims to explore the impact of rs1799963 polymorphism-smoking interaction on the risk of atherosclerosis. For this, control (n = 40) and angiographically confirmed atherosclerotic patients (n = 82) were recruited from District Sargodha, Pakistan. All subjects were genotyped for rs1799963 G/A variants by in-house developed tri-ARMS-PCR assay. Statistical analysis was performed to evaluate the interaction between rs1799963 polymorphism and smoking in relation to atherosclerosis risk. Risk of atherosclerosis was increased by the individual effects of F2 rs1799963 G allele [OR 2.96 (95% CI:1.8-8.08) p = 0.034] and smoking [OR 3.9 (95% CI:1.4-10.8) p = 0.008]. Subjects harboring rs1799963 G allele and who were active smokers had ~ 20 times higher risk of atherosclerosis. Synergy index indicated that combined effect of smoking and rs1799963 G allele was higher than their individual effects, which had a positive interaction with atherosclerosis [synergy index = 2.125 (95% CI: 1.66-2.59)]. These findings suggest a strong interaction between F2 rs1799963 polymorphism and smoking for atherosclerosis. The presence of rs1799963 G allele in conjunction with active smoking status greatly increases the risk of atherosclerosis.