This study examined the effect of collaborative learning (CL) versus traditional lecture-based learning (TL) pedagogies and gender group composition in effecting positive or negative attitudes of biology major and nonmajor men and women students. The experimental research method was administered in experimental and control groups to test the hypotheses. Students’ attitudes refer to their positive or negative feelings and inclinations to learn biology. A nine-factor attitude scale was administered in (1) single-gender nonmajor biology, (2) mixed-gender nonmajor biology, (3) single-gender major biology, and (4) mixed-gender biology major groups. Men (221) and women (219) were randomly assigned into single and mixed-gender classes without groups and single-gender groups (4M) or (4W) and mix-gender (2M+2W) groups. In CL nonmajor and major single-gender groups, women demonstrated significantly higher positive attitudes than men. In contrast, men’s attitudes were significantly improved in mixed-gender CL groups for major and nonmajor sections, and the effect size was larger in mix-gender classes. Women feel less anxious in single-gender groups but more anxious in mixed-gender groups. In mixed-gender groups, men’s self-efficacy, general interest, and motivation enhanced significantly; overall, men experienced greater satisfaction and triggered their desire to collaborate better, affecting all nine attitudinal factors. There was an interaction effect demonstrating the teaching pedagogy’s impact on improving students’ attitudes toward biology; students’ gender and gender-specific group composition have been the most influential factor for nonmajor students. These findings suggest that there is a need for developing gender-specific and context-specific learning pedagogies, and instructors carefully select gender grouping in teaching undergraduate science subjects.