It is important to investigate the influence of incentives and penalties on farmers’ willingness and behavior towards domestic waste separation and the mechanism of influence. It is helpful for the government to formulate policies to guide farmers to actively participate in domestic waste separation, solve the problem of rural domestic waste pollution, and improve the effectiveness of rural waste separation management. By using the public data of 2020CLES, we analyzed the influence of incentives and penalties on farmers’ willingness and behavior towards domestic waste separation by using the Probit model, based on the technology acceptance model, using a chain of multiple mediating effects. The results are as follows. Firstly, the net effect of incentives and penalties on farmers’ willingness and behavior to separate household waste was 6.86%, and the net effect of separation behavior was 33.37%. Relatively speaking, the strengthening effect of incentives and penalties on farmers’ domestic waste sorting behavior is stronger than the promotion effect on farmers’ domestic waste sorting intention. Secondly, perceived ease of use and perceived usefulness had significant chain mediating effects, with perceived ease of use having the strongest mediating effect. Thirdly, the incentives and penalties have a stronger effect on promoting the willingness of female farmers, elderly farmers, and farmers with low education to separate domestic waste. Finally, the influence of individual and household characteristics of farmers on their willingness and behavior to separate household waste varies. Accordingly, suggestions for improving the willingness and behavior of farmers to separate domestic waste are proposed in the following aspects: improving the reward and punishment system, making good use of market instruments, and increasing farmers’ awareness level and value recognition of domestic waste separation.

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