The American shad (Alosa sapidissima) is a common anadromous fish species with ecological and economic importance on the east coast of North America. This iteroparous species undergoes an energetically costly upriver spawning migration in spring. To evaluate metabolic changes associated with this migration, we assessed the maximum activity of five metabolic enzymes (citrate synthase (CS), phosphofructokinase (PFK), lactate dehydrogenase (LDH), β-hydroxyacyl coenzyme A dehydrogenase (HOAD), alanine aminotransferase (GPT)) in liver, red muscle and white muscle during upstream migration in two successive years in the Connecticut River. For aerobic capacity (CS), glycolytic capacity (LDH) and utilization of stored lipid and protein energy (HOAD and GPT), there is a general pattern of increasing activity with a subsequent decline at the most upriver sites. Red muscle CS activity increased by as much as 40% during the migration while white muscle CS activity was 120% higher in the river than in the ocean. In contrast, muscle anaerobic capacity, indicated by PFK, was low as fish entered the river and then increased 5-fold at the most upriver sites. White muscle HOAD increased ∼30% while red muscle HOAD and muscle GPT increased as much as 60%. There were interannual and sex-associated differences in enzyme activity during upstream migration and through time at a single location. In some cases interannual differences can be larger than those seen during upriver migration as in the case of red muscle CS where sampling years differed by 125%. These interannual differences may be a result of differing river conditions that affect migratory effort. We have demonstrated that American shad use tissue and sex-specific regulation of enzyme activity during migration and we suggest that American shad metabolically acclimate to upstream migration.