Maritime shipping is vital for commercial trade and well recognized as a main pathway for the spread of non-native species.1 For over a century, the Panama Canal in Central America has played a major role in global trade, connecting the Atlantic and Pacific oceans. Historically, the introduction of species through the Panama Canal has been relatively low, largely due to the existence of a soft barrier—the freshwater Lake Gatun—inside the canal.2,3,4 However, the 2016 expansion of the Panama Canal involved major structural changes to the canal’s lock system, which may have increased the likelihood that more marine fish species and greater numbers of them enter the lake and eventually cross the canal. To test this prediction, we used standardized quantitative comparisons of the fish communities of Lake Gatun, a system with a rich record of biological introductions,5,6 before (2013–2016) and after (2019–2023) the canal expansion. We observed a shift from a freshwater-dominated to a marine-dominated fish community in several areas inside the lake after 2016. The increase in marine organisms in this aquatic corridor may represent a potential invasion in progress, with a greater likelihood of some species eventually passing through the canal and colonizing the opposite ocean. The ecological and evolutionary consequences of these changes are difficult to predict. However, as most of these marine fishes are top predators with wide niche breadths, their colonization of Atlantic and Pacific oceans will likely alter ecological interactions and potentially lead to ecosystem-level changes.