AbstractUrbanization has affected the economy, ecosystem, and human health. In addition to these direct and physical impacts, urbanization is suggested to affect our preferences and valuation with respect to nature, including people’s perceived values of nature and human–nature connectedness (HNC). Perceived values of nature and HNC are essential for constructing and maintaining sustainable cities. However, the relationship between urbanization, HNC, and perceived values of nature has not been sufficiently elucidated. This study aimed to quantitatively examine this relationship and to generate new insights into methods for enhancing people’s perceived values of nature. The study focused on the following research questions: (1) How different is residents’ HNC under varying urbanization levels? (2) How different is the relationship between HNC and the perceived values of nature under varying urbanization levels? This study categorized perceived values of nature into “relational values” and “instrumental values”. A questionnaire was administered to the residential population in three study areas in Nagareyama City, Japan, each representing a rural, urbanizing, and urban area. The data were subjected to factor analysis, structural equation modeling (SEM), and multi-group SEM. The results indicate that residents’ HNC is weaker in urbanizing and urban areas than in rural areas. The findings also suggest that HNC positively correlates with the perceived values of nature, regardless of urbanization levels. These results indicate that considering HNC is crucial in nature management to understand people’s value perceptions and conservation attitudes as well as to enhance their value perceptions toward surrounding nature under varying urbanization levels.