Under the background of rapid urbanization, the culture of rural tourism destination is increasingly impacted, and the decline of rural culture has become an indisputable fact. Following the development trend of the rural revitalization strategy and integration of culture and tourism in the new era of China, taking Dongshan Town in Suzhou city as an example, we explored the types, spatial pattern and mechanism of cultural stress in rural tourism destinations by using qualitative research and spatial measurement methods. The results show that: (1) the scale of cultural stress of rural tourism destination constructed by the grounded theory and questionnaire survey contains 23 items and 5 dimensions, namely natural landscape culture stress, settlement landscape culture stress, production and living culture stress, interpersonal culture stress and spiritual ritual culture stress. (2) According to the results of GIS spatial analysis, the rural cultural stress space is mostly distributed along the street and water. Traffic jams are the most common stress symptoms in rural areas at the early stage of tourism development. With the development of tourism, the problems which are closely related to the life of local residents, such as water pollution and price rise, are becoming increasingly prominent. Otherwise, the spatial distribution characteristics of the cultural stress in each dimension are quite different. The general space of rural culture stress shows significant agglomeration, among which the production and living culture is most vulnerable to tourism development and shows symptoms of spatial stress. (3) The rural culture stress space has the core-peripheral pattern with multi-core hierarchical distribution, and its spatial agglomeration degree will be significantly improved with the development of tourism. There is a big difference among the spatial agglomeration characteristics of the cultural stress in each dimension. Among them, the settlement landscape cultural stress has the characteristic of large aggregation and small dispersion. (4) Factors affecting cultural stress of rural tourism destination can be identified into the individual and whole levels. The differences in social attributes and conceptual cognition at the individual level, as well as the basic conditions, social development and policy propaganda at the whole level, play a role in the emergence and development of cultural stress in rural tourism destinations.