In this quasi-experimental research, the effect of the inquiry-based 5E instruction on students' learning of the topic of the main subatomic particles forming the atom as well as on their level of motivation was examined. The sample for the study comprised 45 ninth grade students enrolled in two randomly selected classes at a public high school located in a city center in the Aegean region, in the west of Turkey. The topic of main subatomic particles was taught to the experimental group students (n = 21) by inquiry-based 5E instruction, while the control group students (n = 24) were taught the topic using traditional instruction methods. The length of the study was 3 weeks. An academic achievement test which consisted of four open-ended questions and a chemistry motivation scale which was composed of 5-point Likert-type 30 items were administered to both groups as a pre-test and as a post-test. The data were analyzed using the independent-samples t-test and paired-samples t-test statistical analyses. The results showed that inquiry-based 5E instruction on the subatomic particles forming the atom has a significantly positive impact on academic achievement as compared to traditional instruction. Compared with traditional instruction, it was observed that using the inquiry-based 5E instruction did not produce a higher effect on student motivation. Based on the findings, this study provides suggestions that chemistry teachers, educators, and researchers can benefit from.