Rivers in tropical Australia are largely pristine, and ecosystem connectivity is intact, but water resources in northern Australia are under increasing development pressure. A better understanding of the role and life histories of key migratory species is urgently needed to manage the risks posed by development. We investigated the life history of Macrobrachium spinipes (Schenkel, 1902) in the Daly River of the Northern Territory, Australia. We carried out a 13-mo trapping program (2011–2012) at 7 sites along the river between 92 and 405 km from the estuary. We provide the first detailed account of spatial and temporal changes in relative abundance and reproduction, and present information on length at first maturity and Fulton’s body condition factor. Reproduction was strongly seasonal and was restricted to the wet-season months of November to April, followed by a recruitment pulse 3 to 4 mo later during the early dry season. Length at first maturity declined as the reproductive season progressed but did not vary significantly with distance from the estuary. Condition was temporally variable and peaked for both sexes immediately after the wet season before declining throughout the dry season. Relative abundance patterns followed a similar pattern. Contrary to results for other Macrobrachium species in rivers of comparable length, we found an increase in the relative abundance of females throughout the river’s length during the reproductive season and reproductive effort occurring far upstream, over a restricted time period. This result highlights the importance of maintaining connectivity for reproductive success of this species and further recruitment into these upstream reaches.