This paper reports university students' perceptions of Content-Based Instruction (CBI) on English language teaching (ELT) and their relevance to their motivation in terms of L2 motivational self system (Dornyei, 2005). This CBI course was taught at a teacher-training university in the 2013 fall semester. The previous study (Miyasako, under review) examined the students' perceptions of CBI on ELT, extracted three CBI factors (effective, approving, and English-use factors), and revealed that these factors were not much affected by their content understanding and English proficiency. The present study examined: (a) the participants' L2 learning motivation; (b) relationships between their perceptions of CBI on ELT and L2 learning motivation; and (c) effects of their content understanding and English proficiency on L2 learning motivation. The following results were shown. First, the participants' L2 learning motivation centered on ideal L2 self and attitudes to learning English. Second, these two motivational factors (MFs) could explain about 30% of their perceptions of CBI on ELT. Third, MFs relevant to their English proficiency and content understanding are positively ideal L2 self and promotion-focused instrumentality, and negatively ought-to L2 self and prevention-focused instrumentality.