Balancing food production and biodiversity conservation is a big challenge around the world. Eco-friendly agriculture has the potential to overcome these challenges and achieve sustainability. Although some studies show the high valuation of flagship species (e.g., attractive birds and fish) in paddy land where eco-friendly rice is cultivated, limited research has been performed concerning non-specific species such as generalists inhabiting rice paddies that also contribute to agro-biodiversity and ecosystem services. Therefore, this study assesses the valuation of non-flagship vertebrates and invertebrates by applying a choice experiment to vertebrate- and invertebrate-friendly rice. To understand the spatial/regional heterogeneity of the valuation, a questionnaire survey was distributed to both urban and rural citizens in Japan. Our results demonstrated that almost all respondents expressed a desire to protect both vertebrates and invertebrates, with more appreciation for vertebrates than for invertebrates. The analysis also found regional heterogeneity between urban and rural areas in terms of vertebrate and invertebrate evaluations and purchasing intentions. Our findings indicate marketing potential in Japan to promote eco-friendly rice production in relation to vertebrate and invertebrate conservation.