This study aims to evaluate whether incorporating gender differences in portion sizes as part of quantifying a food frequency questionnaire influences the association of total energy intake with mortality. The analysis included 156,434 participants (70,142 men and 86,292 women) in the Multiethnic Cohort Study, aged 45–75 years at baseline. A total of 49,728 deaths were identified during an average follow-up of 18.1 years. Total energy intake and percentage energy from macronutrients were calculated using original portion sizes (PSs) and gender specific (GS)-PS and were divided into quintiles for men and women. The associations of total energy intake and percentage energy from macronutrients with all-cause, cardiovascular disease (CVD), and cancer mortality were examined using Cox regression with adjustment for potential confounders. Mean ± standard deviation daily total energy intake using original-PS was 2449 ± 1135 kcal for men and 1979 ± 962 kcal for women; using GS-PS was 1996 ± 884 kcal for men and 1595 ± 731 kcal for women. For men, the hazard ratios (HRs) (95% confidence intervals) for all-cause, CVD, and cancer comparing the highest to the lowest quintile of total energy intake were 1.05 (1.00–1.10), 1.07 (0.99–1.16), 1.03 (0.95–1.13) using original-PS and 1.07 (1.02–1.12), 1.11 (1.03–1.20), 1.02 (0.94–1.12) using GS-PS, respectively. For women, the corresponding HRs were 1.03 (0.98–1.09), 0.99 (0.91–1.08), 1.10 (1.00–1.21) using original-PS and 1.06 (1.01–1.12), 1.02 (0.94–1.12), 1.07 (0.97–1.18) using GS-PS. Both versions of percentage energy from total fat were associated with an increased risk of all-cause, CVD, and cancer mortality; on the other hand, both versions of percentage energy from carbohydrate showed inverse associations with all-cause, CVD, and cancer mortality in both men and women. When using original-PS and GS-PS, the estimated total energy intake differed, resulting in marginal differences in the associations of total energy intake with all-cause, CVD, and cancer mortality.