The objective of this research is to reveal the influence of business knowledge and vacabulary mastery on the students’ reading comprehension ability of business English. The research was held at the University of Sebelas Maret Surakarta (UNS) and at the Universitas of Muhammadiyah Surakarta (UMS). This was an ex-post facto research with a simple factorial design 2x2 as the research design. The population were all of the fifth semester students of the English Departement of UNS and UMS. The number of the sample was 60 students taken by using a simple random sampling technique The results of the research show that: (1) there is a significant difference in business English reading comprehension ability between the group of students having high vocabulary mastery and those having low vacabulary mastery, (2) there is a significant difference in business English reading comprehension ability between the students having high vocabulary mastery and those having low vocabulary mastery at the student group of high business knowledge, (3) there is no difference in business English reading comprehension ability between the students having high vocabulary mastery and those having low vocabulary mastery at the student group of low business knowledge, (4) Furthermore, it is known that there is an interaction effect between business knowledge and vocabulary mastery on reading comprehension ability of business English. Based on the result of the research, it is concluded that reading comprehension ability of business English can be increased through the improvement of business knowledge and vocabulary mastery.