Purpose: The demand for organ donation is progressively increasing in Malaysia. Healthcare personnel play a crucial role in educating potential donors. This study is aimed to determine the level of knowledge, attitude and practice (KAP) regarding organ donation and associated factors among healthcare workers in UKMMC. Patients and methods: A cross-sectional study was conducted among 255 healthcare workers in UKMMC. Subjects were divided into three groups: doctors, nurses and health assistants. There were 85 participants in each group. A validated 39-item contain self-administered questionnaire was used to assess participants' level of knowledge, attitude and practice regarding organ donation. Results: The mean score is highest among doctors as compared to nurses and health assistants respectively in terms of knowledge (121.74 (84.0%) +/- 12.19; 115.32 (79.5%) +/- 14.16; 109.71 (75.7%) +/- 13.63, p <0.001), attitude (50.27 (83.8%) +/- 5.70; 43.00 (71.7%) +/- 6.88; 42.34 (70.6%) +/- 6.85, p <0.001) and practice (6.33 (63.3%) +/- 1.75; 4.33 (43.3%) +/- 2.19; 3.33 (33.3%) +/- 2.31, p <0.001). Further analysis showed the attitude and practice towards organ donation were significantly higher among doctors (p=0.001), those with tertiary education (p=0.001), males (p=0.001), the Indian ethnicity (p=0.001), Hindus (p=0.001) and those with a high monthly income (>RM10000) (p=0.001). However, knowledge was significantly associated with younger age (21-30) (p=0.026), being Chinese (p=0.034) and having a moderate monthly income (RM500010000) (p=0.001). Conclusion: The factors which affect attitude and practice are not associated with knowledge. Despite good knowledge, willingness to donate is low when attitude is poor. An organ donation campaign should be conducted to increase attitude and practice among those who have knowledge but are not willing to practise.