Biology education, as a part of science education in Ethiopia, continues to face persistent challenges characterized by low student achievement, limited classroom engagement, and unfavorable attitudes toward the subject. These challenges are often associated with the disconnection between formal science instruction and learners‘ sociocultural experiences. Guided by culturally responsive pedagogy and social constructivism, this study examined the effect of integrating Indigenous Knowledge (IK) into Grade 8 Biology instruction in Injibara City, Ethiopia. Specifically, the study investigated the effects of IK-based instruction on students‘ academic achievement, engagement, and attitudes toward ecology, while also examining whether gender moderated these effects. The study employed a mixed-methods approach using a quasi-experimental non-equivalent pretest–posttest control group design. Participants were assigned to either an experimental group receiving IK-integrated instruction or a control group taught using conventional methods over a six-week instructional period. Quantitative data were collected using the ecology achievement Test (EAT), ecology attitude questionnaire (EAQ), and ecology engagement questionnaire (EEQ), while qualitative data were generated through focus group discussions (FGDs) and classroom observations. Quantitative data were analyzed using independent samples t-tests and Analysis of Covariance (ANCOVA), whereas qualitative data were analyzed thematically. The findings revealed statistically significant differences in favor of the experimental group across all measured outcomes. After controlling for pretest scores, students exposed to IK-based instruction achieved significantly higher posttest scores in ecology achievement, engagement, and attitude compared to those taught through conventional instruction (p < .05). The magnitude of these differences ranged from moderate to large effect sizes, indicating substantial practical significance of the intervention in improving students‘ learning outcomes. The ANCOVA results further demonstrated that the instructional intervention accounted for a meaningful proportion of variance in posttest performance, suggesting that the observed improvements were educationally important rather than only statistically significant. No statistically significant interaction effects were found between instructional approach and gender, indicating that the effectiveness of IK- based instruction was comparable for both male and female students and therefore pedagogically gender-inclusive. Qualitative findings supported the quantitative results. Students consistently perceived the instructional approach as making ecological concepts more understandable, meaningful, and connected to their daily lives. The study concludes that integrating IK into