Catering for learner diversity is one of the key areas in the recent educational reform in Hong Kong. Pre-service teacher education acts as a fundamental way to equip pre-service teachers ready for accommodating diverse learning needs and to build up pre-service teachers' self-efficacy. The purpose of the study is to examine prospective teachers' teaching beliefs toward differentiated instruction and teaching efficacy. Using a sequential mixed methods design that contains a questionnaire, focus group interviews, and individual interviews, prospective teachers undertaking a 13-session course regarding differentiated instruction as offered by a local university in Hong Kong participated in the study from January to April 2013. Changes in teaching beliefs regarding differentiated instruction as well as teaching efficacy levels were found. More positive attitudes toward differentiated teaching were found; however, there existed different concerns including class management and conflicts with personal teaching beliefs. These concerns may be related to practical experiences and confidence as well as expectations upon students. Implications for future course development and research are discussed at the end of the paper.