Humulus lupulus (hop) is a necessary material for beer brewing. Improved breeding cultivars of hops with enhanced tolerance to environmental stresses, such as drought and heat stress, accompanying climate change have been developed. However, a propagation system, which is needed for the proliferation of new cultivars, is not currently available for hops. In this study, we found that treatment of stem explants with 0.01–0.05 ppm gibberellic acid (GA₃) induced the development of axillary buds in the hop cultivar Kirin-2, resulting in the proliferation of shoot branching. Additionally, 0.01 ppm benzyl adenine (BA) enhanced the development of axillary buds formed in response to 0.05 ppm GA₃ in various hop cultivars, particularly Nugget. The development of axillary buds was strongly repressed by the application of 0.05 ppm BA at a concentration equal to the 0.05 ppm GA₃ concentration, which showed the possibility that a high concentration of cytokinin preferentially prevents the effect of GA₃ on the development of axillary buds in hops. These results indicated that combined treatment of stem explants with GA₃ and cytokinin at appropriate concentrations is effective for the propagation of proliferated hop cultivars through shoot branching.