The purpose of this study was to examine the effect of the web-based learning program integrating learning motivation on improving learning motivation in biology class. It was designed for science education considering learning motivation strategy, blended learning strategy and mutual interaction strategy. Subjects were totally 187 middle school students, who consisted of experimental group 95 (three classes) and control group 92 (three classes). To find out the change of learning motivation, they were divided into three level subgroups; high, middle and low motivational level. As a result, the experimental group had higher in every area of learning motivation elements (attention, relevance, confidence, and satisfaction) than those of the control group (p<.05). In particular, those of students who were in the low-level motivation were improved significantly. The results suggested that the web-based learning program integrating learning motivation should be effective for improving learning motivation of middle school students in biology class.