OBJECTIVES: This study aims to assess the Knowledge, Attitude and Practice (KAP) regarding blood donation among health care workers in Malaysia.  METHODS: A cross-sectional survey was conducted among153 health care workers enrolled randomly from 2 district hospital in Malaysia.  RESULTS: Out of 153 participants, 76 (49.5%) reported to donate blood at least once in their life time.  Among the demographic characteristics only female gender was significantly associated (p=0.046) with blood donation practice where female reported to donate less blood than male. Thirty seven (24.5%) of the respondents assumed that blood donation carries risks of infections. Knowledge about blood donation frequency per year was known to 46 (30%).  In this study 108(70.6%) responded reported the correct maximum age for blood donation. Knowledge regarding the minimum gap between two blood donations was not known to 47(31%) of the participants. Participants lacked correct knowledge about amount of blood collected in one donation 19 (12.4%). Low knowledge scores were noted for the number of days in which blood volume returns to normal after donation where 130(85%) had no knowledge. Most of the respondent 120(78.4%) reported that >5% of population donate blood in Malaysia. The most common reason of donation was due to calls for blood donation from friends and relatives 40(52.6%). The most common reasons of not donating blood was given as unfit for blood donation 54(70.1%) followed by fear of needles 43(55.8). Most of the participants agreed that government should offer better incentives to improve blood donation practices. CONCLUSIONS: Donation of blood among health care workers in Malaysia needs education and motivation through dissemination of information regarding blood donation by conducting awareness programs, courses, seminars, pamphlets, posters and public activities especially through the electronic media.