Background: During isolation, physical activity can enhance residents’ well-being. The objective of the present study was to examine the associations between residents’ physical activity, health values, and well-being during isolation.Methods: Based on the physical activity rating scale, health values scale, subjective well-being scale, and the satisfaction with life scale, 505 valid questionnaires were collected online from 31 provinces, municipalities and autonomous regions in China. A series of multiple linear regression models were established to study the relationship between variables, and the bootstrap confidence interval was selected to test the mediating effect.Results: The results showed that during the period of isolation, physical activity directly(b =0.463, p < 0.001) or indirectly(b =0.358, p < 0.001) had a positive impact on residents’ well-being through the mediating effect of health values. There is a positive correlation between physical activity and health values(b =0.710, p < 0.001), while health values had a direct positive impact on well-being(b =0.504, p < 0.001). In addition, moderate amount of physical activity is more likely to improve the well-being of residents during home isolation compared to small and large amount of physical activity.Conclusions: Moderate exercise at home and regular physical activity are beneficial to our physical and mental health, especially to improve the overall well-being. At the same time, it is necessary for the World Health Organization and governments at all levels to strengthen the guidance and publicity of health values, so as to eliminate the impact of COVID-19 on people to a greater extent.