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Abstract The brain is organized into intrinsically connected functional networks that can be reliably identified during resting-state functional magnetic resonance imaging (fMRI). Healthy aging is marked by decreased network segregation, which is linked to worse cognitive functioning, but aging-related changes in emotion are less well characterized. Valence bias, which represents the tendency to interpret emotionally ambiguous information as positive or negative, is more positive in older than younger adults and is associated with differences in task-based fMRI activation in the amygdala, prefrontal cortex, and a cingulo-opercular (CO) network. Here, we examined valence bias, age, and resting-state network segregation of 12 brain networks in a sample of 221 healthy individuals from 6 to 80 years old. Resting-state network segregation decreased linearly with increasing age, extending prior reports of de-differentiation across the lifespan. Critically, a more positive valence bias was related to lower segregation of the default mode network (DMN), due to stronger functional connectivity of the DMN with CO and, to a lesser extent, the ventral attention network (VAN) in all participants. In contrast to this overall segregation effect, in participants over 39 years old (who tend to show a positive valence bias), bias was also related to weaker connectivity between the DMN and Reward networks. The present findings indicate that specific interactions between the DMN, a task control network (CO), an emotion processing network (Reward), and, to a weaker extent, an attention network (VAN), support a more positive valence bias, perhaps through regulatory control of self-referential processing and reduced emotional reactivity in aging. The current work offers further insight into the functional brain network alterations that may contribute to affective well-being and dysfunction across the lifespan.