Drawing on social exchange and self-determination theories, we tested a model in which self-determined motivation acts as a mediating factor in the relationship of organizational characteristics (perceived organizational support) and work attitudes (affective commitment) to organizational citizenship behaviors (OCBs). A total of 590 nurses from two Italian hospitals participated in the study. A sample of 327 nurses was used to verify the mediating role of intrinsic motivation (Hypothesis 1). To show that the mediation effect only operates for intrinsic motivation, another sample of 263 nurses was used to test the absence of a mediating role of extrinsic motivation (Hypothesis 2). The results indicated that affective commitment and perceived organizational support (POS) were differently and directly related to citizenship behaviors, and indirectly via intrinsic motivation, which was a significant mediating variable. In fact, the results from Hypothesis 2 showed that extrinsic motivation did not mediate the relationship between either affective commitment or POS, and OCBs, thus further supporting Hypothesis 1. The findings of this study have important implications for human resource management strategies, which could increase POS, affective commitment, and intrinsic motivation in order to encourage employees to use good citizen behaviors.