In today's business climate, organizational knowledge has been widely accepted as a principle organizational source for sustainable advantages for global competitiveness. Inter-organizational knowledge transfer lays a significant foundation for obtaining new organizational knowledge. The role of middle managers in inter-organizational knowledge transfer is getting more and more attentions nowadays, although it cannot be more than enough. In addition, considerable evidence supports the importance of culture in the success or failure of knowledge transfer within organizations. The main purpose of this research is to identify what roles of middle managers play in each stage of inter-organizational knowledge transfer as well as the impact of national culture on such roles. The research used a case study methodology and was conducted among Canadian, American and Chinese middle managers in two well-known multinational organizations. The findings suggest that first, middle managers play the roles of Radar, Filter and Champion in the initiation stage, the role of Coordinator in the interrelation stage and the role of Problem solver in the implementation stage; second, Chinese middle managers are involved less than those from North America in the activity 'Suggesting and prioritizing the different courses of action to acquire new knowledge', 'Defining and justifying the importance of new knowledge transfer proposals to upper-level managers' and 'Embedding the newly acquired knowledge in organizational processes and routines'. This study opens new insights of research in knowledge transfer that link up the roles of middle managers, national culture, and the effectiveness of knowledge transfer.