The aim of this paper is to examine the effects of sequencing instructional materials and learners' prior knowledge on learning ESL (English as a second language) through an online learning course. 121 fifth-grade students from an elementary school in Korea participated in the study. Each participant was allocated to one cell of a 2 × 2 between-subjects factorial design, with an instructional format (i.e. sequential or concurrent) as a first factor, and prior knowledge (i.e. high or low) as a second factor. Instructions of the words and grammar rules were allocated to each page for the sequential group, whereas integrated on a single page for the concurrent group. The result indicates that the sequential instruction of words followed by grammar explanations was more effective for the students with higher levels of prior knowledge than the concurrent presentation. The reverse was also found for the students with lower levels of prior knowledge in English. There are four possible explanations for the results. First, split-attention effect, temporal-contiguity effect, or transient information effect might occur. Second, worked-example effect might occur and over-ride the effect of sequencing. Third, intrinsic and extraneous cognitive load might intertwine each other. Last, the levels of element interactivity in two instructional formats might be different from each other. The implications for instructional designers and future research are discussed.