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Building students' spatial ability to assist them in understanding molecular geometry requires proper multimedia. This study investigated the effectiveness of 3D molecular interactive multimedia (3D-MIM) in improving chemistry students' spatial ability. Thirty chemistry education students of a public university in East Java, Indonesia, participated in this study. This pre-experimental pretest-posttest design employed a set of spatial ability tests referring to the three main dimensions of spatial abilities (spatial relationship, spatial orientation, spatial visualization) and semi-structured interview. Our finding showed that students' spatial abilities for the three domains after experiencing the 3D-MIM fall in the strong and large effects category. The values of N-gain falling in the moderate category and effect size (d) in the high category confirm the effectiveness of the 3D-MIM.