Eudaimonic well-being (EWB) is the fulfillment of human potential and a meaningful life. Previous studies have shown that personality traits, especially extraversion, neuroticism, and conscientiousness, significantly contribute to EWB. However, the neurobiological pathways linking personality and EWB are not understood. Here, we used resting-state functional magnetic resonance imaging (rs-fMRI) to investigate this issue. Specifically, we correlated individuals' EWB scores with the regional fractional amplitude of low-frequency fluctuations (fALFF) of the brain, and then examined how personality traits predicted EWB-related spontaneous brain activity. We found that EWB was positively correlated with the fALFF in the right posterior superior temporal gyrus (pSTG) and thalamus, and negatively correlated with the strength of the thalamic-insular connectivity. More importantly, we found that personality traits influenced EWB in different ways. At the regional level, the fALFF in the pSTG and thalamus mediated the effects of neuroticism and extraversion on EWB, whereas the thalamus mediated the effect of conscientiousness on EWB. At the functional connectivity level, the thalamic-insular connectivity only mediated the effect of neuroticism on EWB. Taken together, our study provides the first evidence that EWB is associated with personality traits through different neural substrates.