In China, adolescents are frequently left behind. To date, few studies have focused on the pro-social tendencies of left-behind adolescents and the relationship of family function, self-esteem, and pro-social tendency is yet to be examined. This study, therefore, aims to understand the status of pro-social tendency of left-behind adolescents and to explore the mediating and moderating roles of self-esteem in the relationship between family function and pro-social tendency. A large, school-based survey was conducted in three Chinese provinces. An analysis of covariance was first used to identify the differences in pro-social tendency between adolescents who were and were not left behind. We then analyzed the variance within left-behind adolescents using demographics, left-behind type, years of being left-behind, and caregiver related characteristics. A structural equation model was used to analyze the relationship of family function, self-esteem, and pro-social tendency, with bootstrapping used to explore the mediating role of self-esteem. Additionally, an ordinary least squares regression was used to examine the moderating effect of self-esteem. The results showed that the pro-social tendency of left-behind adolescents was lower than in non-left-behind adolescents (F = 15.11, p = 0.0001). Family function was positive related to pro-social tendency (r = 0.259), which had not only a direct effect on pro-social tendency (β = 0.254), but also an indirect effect through self-esteem (β = 0.071, bias-corrected 95% CI: 0.051:0.090; percentile 95% CI: 0.053:0.092). Additionally, 21.85% of the total effect of family function on pro-social tendency was mediated by self-esteem. Furthermore, self-esteem negatively moderated the relationship between family function and pro-social tendency (β = -0.208, p < 0.0001), such that the effect of family function on pro-social tendency became weaker as self-esteem increased. The current study verified the negative effect of being left behind on the social development of adolescents and contributed to the understanding of the importance of self-esteem in the relationship between family function and pro-social tendency. Interventions aimed at enhancing self-esteem should be developed and implemented in left-behind adolescents to promote wellness in the entirety of psychological and social outcomes.