Grit is a personality trait, conceptualized as perseverance of effort and consistency of interests in long-term goals. Previous research has shown that grit is associated with various positive outcomes, including well-being. Despite extensive research on grit, most studies relied on self-reported measures rather than objective measures. To address this gap, our study investigated the relationship between grit and physiological responses, focusing on resting-state heart rate variability (HRV)-a physiological marker of well-being and adaptability. Additionally, we examined whether this relationship was unique to grit and not explained by other related psychological constructs (i.e., conscientiousness and self-control). A total of 206 healthy college students participated in this study (M_age = 21.03, SD = 2.48, age range = 18-33; 111 women). Results of hierarchical regression analyses showed that grit significantly predicted resting-state HRV measures (i.e., RMSSD, SDNN, and HF) even after controlling for conscientiousness, self-control, age, gender, and respiration rate. These findings suggest that grit may play an important role in maintaining improved biological responses in daily life, beyond the effects of neighboring constructs.