Humic acid has been considered as one of the most significant sources in feed water causing organic fouling of reverse osmosis (RO) membranes, but the relationship between the fouling behavior of humic acid and the change of its molecular structure has not been well developed yet. In this study, the RO membrane fouling behavior of humic acid was studied systematically with ozonation as a pretreatment method to control RO membrane fouling. Furthermore, the effect of ozone on the structure of humic acid was also explored to reveal the mechanisms. Humic acid alone (1090 mg/L, in deionized water) was found not to cause obvious RO membrane fouling in 45-h operation. However, the presence of Ca2+ aggravated significantly the RO membrane fouling caused by humic acid, with significant flux reduction and denser fouling layer on RO membrane, as it was observed by scanning electron microscope (SEM) and atomic force microscope (AFM). However, after the pretreatment by ozone, the influence of Ca2+ was almost eliminated. Further analysis revealed that the addition of Ca2+ increased the particle size of humic acid solution significantly, while ozonation reduced the SUVA254, particle size and molecular weight of the complexes of humic acid and Ca2+ (HA-Ca2+ complexes). According to these results and literature, the bridge effect of Ca2+ aggregating humic acid molecules and the cleavage effect of ozone breaking HA-Ca2+ complexes were summarized. The change of the structure of humic acid under the effect of Ca2+ and ozone is closely related to the change of its membrane fouling behavior.