The aim of this study was to investigate the relationship between human resource management (HRM) practices and organizational performance on the basis of 247 valid and reliable questionnaires distributed to managers at different management levels working in Jordanian manufacturing firms. The study also aimed to explore the mediating role of knowledge management as well as the moderating effect of organizational culture on the relationship between HRM practices and organizational performance. Ten HRM practices and 10 indicators of organizational performance were adopted for the purpose of this study. Knowledge management was measured by examining three processes; knowledge creation, sharing and utilization. Organizational culture was measured according to passive/defensive, aggressive/defensive and constructive cultures. The results of the study supported the presumed hypotheses. Hence, HRM practices significantly predicted organizational performance. Knowledge management mediated the relationship between HRM practices and organizational performance. Finally, it was found that organizational culture moderated the relationship between HRM practices and organizational performance as well as the relationship between HRM practices and knowledge management. Constructive cultures play a positive role in the relationship between HRM practices and organizational performance (OP), while defensive cultures negatively affect the relationship between HRM practices and knowledge management (KM). The main contribution of this study to the literature on HRM, KM and OP derives from the lack of prior studies addressing the same purposes as this study. The study informs researchers and managers that both knowledge management and organizational culture mediate and moderate the impact of HRM practices on organizational performance to a considerable extent.