Background Monounsaturated fatty acids (MUFAs) improve blood lipid profiles in intervention studies, but prospective evidence with regard to MUFA intake and coronary heart disease (CHD) risk is limited and controversial. Objective We investigated the associations of cis MUFA intake from plant (MUFA-P) and animal (MUFA-A) sources with CHD risk separately among 63,442 women from the Nurses' Health Study (1990-2012) and 29,942 men from the Health Professionals Follow-Up Study (1990-2012). Design Intakes of MUFA-Ps and MUFA-As were calculated by using validated food-frequency questionnaires collected every 4 y. Incident nonfatal myocardial infarction and fatal CHD cases (n = 4419) were confirmed by medical record review. Results During follow-up, MUFA-Ps and MUFA-As contributed 5.8-7.9% and 4.2-5.4% of energy on average, respectively. When MUFA-Ps were modeled to isocalorically replace other macronutrients, HRs (95% CIs) of CHD were 0.83 (0.68, 1.00) for saturated fatty acids (SFAs; 5% of energy), 0.86 (0.76, 0.97) for refined carbohydrates (5% of energy), and 0.80 (0.70, 0.91) for trans fats (2% of energy) (P = 0.05, 0.01, and 0.001, respectively). For MUFA-As, corresponding HRs (95% CIs) for the same isocaloric substitutions were 1.04 (0.79, 1.38) for SFAs, 1.11 (0.91, 1.35) for refined carbohydrates, and 0.88 (0.77, 1.01) for trans fats (P = 0.76, 0.31, and 0.08, respectively). Given the common food sources of SFAs and MUFA-As (Spearman correlation coefficients of 0.81-0.83 between these groups of fatty acids), we further estimated CHD risk when the sum of MUFA-As and SFAs (5% of energy) was replaced by MUFA-Ps, and found that the HR was 0.81 (95% CI: 0.73, 0.90; P < 0.001) for this replacement. Conclusions The largely different associations of MUFA-Ps and MUFA-As with CHD risk suggest that plant-based foods are the preferable sources of MUFAs for CHD prevention. These findings are observational and warrant confirmation in intervention settings. This study was registered at clinicaltrials.gov as NCT00005152 and NCT00005182.