Colloidal storage haze is one of the principal indicators of beer quality. In this study, the changes of malt hordeins during the brewing process and the roles of malt hordeins in the formation of beer haze were investigated using two-dimensional electrophoresis (2-DE). It was found that most of malt hordeins disappeared during the brewing process and only the few faint hordein spots (about eight spots) were detected in beer haze. Moreover, these spots were found in wort protein and beer haze protein. The complex 2-DE patterns of malt hordein, wort and beer haze displayed a relationship with some chemical modifications during the brewing. These spots were analyzed and identified as B and γ3 hordein and storage protein (Hordeum vulgare) by mass spectrometry. Our results suggested that hordein might be minor haze-active proteins in beer but was a critical factor in the formation of beer haze.