Background and purpose: Despite sharing some metabolic and pathological mechanisms, the reported association between total homocysteine (tHcy), asymmetric dimethylarginine (ADMA) and stroke remains controversial, particularly in Hispanic populations from developing countries in which genetic, socioeconomic, and nutritional factors are different to those described in developed countries. Our objectives were to determine the relationships of these factors to stroke and to each other independent of other cardiovascular risk factors, and to explore potential sex differences. Methods: This national (Colombia) multicenter case–control study included 238 cases and 238 controls to evaluate traditional and emerging risk factors for ischemic stroke including tHcy and ADMA plasma levels. Results: The median plasma levels of tHcy were 8.48 μM for controls and 10.01 μM for cases ( P < 0.0001). Plasma levels of tHcy between 12 and 50 μM were considered moderate hyperhomocysteinemia (HtHcy). There were no differences in plasma ADMA concentration between groups ( P = 0.40). Plasma levels of ADMA and creatinine were not correlated ( P = 0.47). After adjusting for confounding factors, the presence of HtHcy was strongly associated with stroke (OR 8.97; P < 0.0001). The adjusted association between HtHcy and stroke in men (OR 9.98) was comparable to that in women (OR 8.98) ( P = 0.41). Conclusions: In this Hispanic population, with relatively normal renal function, plasma levels of tHcy but not ADMA were associated with stroke independent of other cardiovascular risk factors.