Streptococcus iniae is a zoonotic pathogen and one of the major aetiologic agents of streptococcosis. In White Sturgeon Acipenser transmontanus, S. iniae infection typically presents as a necrotizing and heterophilic myositis, causing 30–50% mortality in infected fish. To gain a better understanding of the pathogenesis of streptococcosis in White Sturgeon, and to identify the experimental route of infection that most closely mimics the natural disease, fingerlings were challenged with a single dose of 1.3 × 108 cells/fish of S. iniae that was administered via intracoelomic/intraperitoneal (IC) or intramuscular (IM) routes. Acute mortalities were present only in the IM‐challenged fish, with first mortality occurring 4 d postchallenge and the mortality rate reaching 18.3% after 9 d. The challenged fish presented erratic swimming, ulcerative skin lesions, and hemorrhages in the liver and swim bladder. Streptococcus iniae was recovered from the kidney and brain tissues of moribund and dead fish. Histopathologic analysis of fish that died acutely revealed massive proliferation of bacteria in the muscle at the injection site and within vascular organs such as the heart and spleen, with variable amounts of tissue necrosis including a necrotizing myositis. Fish that died closer to 9 d postchallenge demonstrated more pronounced multifocal to locally extensive granulomatous inflammation of skeletal muscle at the injection site, liver, kidney, and spleen. No mortality, clinical signs, or gross changes were observed in the control or IC‐challenged fish. Postmortem evaluation of 10 survivors in each treatment was performed to determine carrier status in the brain and posterior kidney tissues. The prevalence of S. iniae in survivors was 10% and 0% in the IM‐ and IC‐challenged groups, respectively. The results from this study suggest that IM‐injection challenge methods are suitable for inducing streptococcosis in White Sturgeon, and they may be the preferred method for studying the pathogenesis of the naturally occurring disease in this species.