As individuals age, the entorhinal cortex (ERC) and hippocampus-crucial structures for memory-tend to atrophy, with related cognitive decline. Simultaneously, lifestyle factors that can be modified, such as exercise and sleep, have been separately linked to slowing of brain atrophy and functional decline. However, the synergistic impact of fitness and sleep on susceptible brain structures in aging adults remains uncertain. We examined both independent and interactive associations of fitness and subjective sleep quality with regard to ERC thickness and hippocampal volume in 598 middle-aged and older adults from the Human Connectome Lifespan Aging Project. Cardiorespiratory fitness was assessed using the 2-min walk test, whereas subjective sleep quality was measured with the continuous Pittsburgh Sleep Quality Index global score. High-resolution structural magnetic resonance imaging was used to examine mean ERC thickness and bilateral hippocampal volume. Through multiple linear regression analyses, we investigated the moderating effects of subjective sleep quality on the association between fitness and brain structure, accounting for age, sex, education, body mass index, gait speed, and subjective physical activity. We found that greater cardiorespiratory fitness, but not subjective sleep quality, was positively associated with bilateral hippocampal volume and ERC thickness. Notably, significant interaction effects suggest that poor subjective sleep quality was associated with a weaker association between fitness and both hippocampal volume and ERC thickness. Findings suggest the potential importance of both cardiorespiratory fitness and subjective sleep quality in preserving critical, age-vulnerable brain structures. Interventions targeting brain health should consider potential combined effects of sleep and fitness on brain health.