To identify gender difference in safe and unsafe practice of pesticide handling in tobacco farmers of Malaysia, we conducted a 20-item questionnaire interview on storage of pesticide (4 questions), mixing of pesticide (3 questions), use of personal protective equipment and clothing while spraying pesticide (7 questions), activities during and after spraying of pesticide (5 questions), and maintenance of pesticide sprayer (1 question) in 496 tobacco farmers (395 males and 101 females) in Bachok District, Kelantan, Malaysia. Duration of employment was significantly longer in females than those in males (p<0.001). In addition, proportion with no formal education in females was significantly higher than those in males (p<0.05). The following eight common factors were extracted from the 20 questionnaires by principal components factor analysis after varimax rotation in all farmers: (1) use of personal protective equipment, (2) unsafe work habit, (3) reading and following instructions on pesticide label, (4) security, storage and disposal of pesticide container, (5) safe work habit, (6) proper handling of pesticide and maintenance of pesticide sprayer, (7) use of personal protective clothing, and (8) safe handling of pesticide. Results of analysis of covariance for the eight factor scores of all male and female farmers, controlling for educational level and duration of employment, showed that: (1) factor scores for use of personal protective equipment (p<0.001), use of personal protective clothing (p<0.001) and safe work habit (p<0.001) in females were significantly lower than those in males; (2) conversely, factor scores for reading and following instruction on pesticide label (p<0.001) and proper handling of pesticide and maintenance of pesticide sprayer (p<0.01) in males were significantly lower than those in females; and (3) there were no significant differences in other three factor scores (p>0.05). We therefore conclude that: (1) for female tobacco farmers, choice of personal attire tend to result in lower scores on use of personal protective equipment and personal protective clothing while personal hygiene practices result in lower score on safe work habit; and, (2) for male tobacco farmers, the lower scores on reading and following instruction on pesticide label and mixing pesticide and maintenance of pesticide sprayer in good condition suggests that they were not primarily involved in these activities. It is postulated that these differences in safe and unsafe practices of pesticide handling across gender is related to the choice of personal attire, personal hygiene practices and division of labour within farming households which in turn is influenced by prevailing sociocultural norms in the community.