Rationale: Dietary monounsaturated fatty acids (MUFAs) can come from both plant and animal sources with divergent nutrient profiles that may potentially obscure the associations of total MUFAs with chronic diseases. Objective: To investigate the associations of cis -MUFA intake from plant (MUFA-P) and animal (MUFA-A) sources with total and cause-specific mortality. Methods and Results: We followed 63 412 women from the NHS (Nurses’ Health Study; 1990–2012) and 29 966 men from the HPFS (Health Professionals Follow-Up Study; 1990–2012). MUFA-Ps and MUFA-As were calculated based on data collected through validated food frequency questionnaires administered every 4 years and updated food composition databases. During 1 896 864 person-years of follow-up, 20 672 deaths occurred. Total MUFAs and MUFA-Ps were inversely associated with total mortality after adjusting for potential confounders, whereas MUFA-As were associated with higher mortality. When MUFA-Ps were modeled to isocalorically replace other macronutrients, hazard ratios (HRs, 95% CIs) of total mortality were 0.84 (0.77–0.92; P <0.001) for replacing saturated fatty acids, 5% of energy); 0.86 (0.82–0.91; P <0.001) for replacing refined carbohydrates (5% energy); 0.91 (0.85–0.97; P <0.001) for replacing trans fats (2% energy), and 0.77 (0.71–0.82; P <0.001) for replacing MUFA-As (5% energy). For isocalorically replacing MUFA-As with MUFA-Ps, HRs (95% CIs) were 0.74 (0.64–0.86; P <0.001) for cardiovascular mortality; 0.73 (0.65–0.82; P <0.001) for cancer mortality, and 0.82 (0.73–0.91; P <0.001) for mortality because of other causes. Conclusions: Higher intake of MUFA-Ps was associated with lower total mortality, and MUFA-As intake was associated with higher mortality. Significantly lower mortality risk was observed when saturated fatty acids, refined carbohydrates, or trans fats were replaced by MUFA-Ps, but not MUFA-As. These data suggest that other constituents in animal foods, such as saturated fatty acids, may confound the associations for MUFAs when they are primarily derived from animal products. More evidence is needed to elucidate the differential associations of MUFA-Ps and MUFA-As with mortality.