This research investigated gender differences and differential item functioning (DIF) on Science parts of the Basic Competence Test for Junior High School Students (BCTEST) from 2001 to 2005 administrations. Results based on overall science performances indicated that there were no visible gender differences among total groups. However, females performed slightly better than males among low-achieving groups (bottom 10%); males performed slightly better than females among high-achieving groups (top 10%). Results based on performance of subjects indicated that females outperformed males on Health Education, males outperformed females on Physics and Earth Science, and there were no visible gender differences on Biology and Chemistry among total groups. In addition, there were no visible gender differences among low-achieving and high-achieving groups with only a few exceptions. The exceptions were that females outperformed males on Health Education among both low-achieving and high-achieving groups, and males outperformed females on Physics among high-achieving groups. In the study of gender DIF, Mantel-Haenszel procedure was used. The results of DIF analyses showed that total 52 (about 9%) of the 575 items across test administrations were categorized as DIF. Among the factors explored in the study, including subject, cognitive demands and types of attachment materials, the subject was the most salient factor that affected the performance of the DIF measures. The DIF items from Health Education, Chemistry and Biology subjects tended to favor females; however, the DIF items from Earth Science and Physics subjects tended to favor males. The follow up review and judgment were conducted for these DIF items, and no construct irrelevant factors were found. That is, though these items displaying DIF, they were not biased items. Implications based on the findings of this study were proposed for educators, test developers and researchers.