Empathy has been regarded by environmental thinkers as a key in conservation efforts. Nevertheless, systematic research on empathy toward nature, particularly from the personality perspective, has been lacking in psychology. The present research thus provides this needed investigation by testing four propositions regarding a new construct—dispositional empathy with nature (DEN), which refers to the dispositional tendency to understand and share the emotional experience of the natural world. In five studies with 817 participants in total (including university students and working adults from two societies), DEN robustly and uniquely predicted conservation behavior (Proposition 1). Females, respondents who felt close to nature, and participants who considered nature to be sentient exhibited stronger DEN (Propositions 2–4). DEN was distinct from empathy with humans and a number of known determinants of conservation behavior (including personality traits, values, emotional involvement with nature, environmental concern, and social desirability bias). Taken together, these findings highlight the possibility of developing a theory of empathy with nature by referring to the existing understanding about empathy with humans. The construct of DEN has much theoretical utility, as it sheds new light on several under-explored issues in conservation psychology (including the gender gap in environmentalism, the role of connection to nature, and the role of anthropomorphism), and bears practical implications for the promotion of environmentalism. In addition, the newly developed scale for DEN is potentially useful for assessing the efficacy of environmental education programs.