This study examined the role of gender on short-term heart rate variability (HRV) and the correlation between subjective ratings of stress and HRV in healthy adults. Standardized short-term HRV measurement and self-administered stress response inventory (SRI) were obtained in 441 healthy women and 1440 healthy men. Hierarchical multiple regressions suggested that there was no gender by stress interaction in explaining HRV. However, there were significant gender differences in the associations between stress and HRV (the standard deviation of the NN interval (SDNN), high frequency (HF), low frequency (LF)/HF (F(1, 1878) = 7.706, p < .01; F(1, 1878) = 29.132, p < .01; F(1, 1878) = 49.685, p < .01). In men, only HF (r = -.56, p = .031) showed such an association; whereas in women, the SRI total scores were negatively correlated with SDNN (r = -.103, p = .032), total power (TP) (r = -.104, p = .030), and HF (r = -.129, p = .007), and positively correlated with LF/HF (r = .111, p = .020) when adjusted for age, alcohol drinking, smoking, and caffeine intake. There are gender differences in the association between psychological stress response and HRV. Gender also showed a significant impact on short-term HRV measurement. Given that both clinicians and researchers are increasingly relying on HRV assessment, our work suggest that gender based norms are very important.