Anthropomorphism of nature can promote pro-environmental behavior (PEB). However, its underlying mechanism and these age-related changes are unclear. We propose that connectedness to nature and environmental guilt mediate the relationship between anthropomorphism of nature and PEB. The present study tests the hypotheses based on a cross-sectional sample of 1364 residents aged 15-76 years, using structural equation modeling. We found that: (1) environmental guilt decreases, but PEB increases, with age; (2) anthropomorphism of nature decreases in early adulthood and increases in old age; (3) connectedness to nature decreases in mid-late adolescence and increases in early adulthood. Connectedness to nature and environmental guilt have a serial mediating effect in the relationship between anthropomorphism of nature and PEB, with cross-age stability. These findings contribute to enriching the understanding of PEB from the human and nature perspective, and enhancing anthropomorphism of nature that could promote PEB in residents at different ages, through connectedness to nature and environmental guilt.