Endosymbiosis – where one species lives inside another – underpins the evolution of complex life and the function of multiple diverse ecosystems, including those important for health and food systems. Yet the mechanisms that make associations stable, and thus determine their persistence, are poorly understood. Fitness trade-offs between contrasting intracellular and extracellular niches could act to stabilise endosymbioses because adaptation to either niche is predicted to reduce fitness in the alternate niche, thus reinforcing symbiosis. We experimentally evolved four diverse Chlorella green algal endosymbionts of Paramecium bursaria to free-living conditions supplying either an amino acid, as provisioned intracellularly by hosts, or nitrate, as available in freshwater, as the sole nitrogen source. Experimental algal populations adapted to free-living environments generally increased population densities over time. In one experimental algal strain, adaptation to the nitrate free-living environment, but not the amino acid environments, drove the loss of symbiotic benefits to P. bursaria. Genome sequencing of evolved algal lines revealed genomic divergence between nitrate-adapted and amino acid-adapted lines in variants predicted to affect carbon and/or nitrogen metabolism, suggesting a metabolic trade-off may underlie the loss of beneficial symbiosis. We are currently analysing the results of untargeted metabolomics using DESI-MS to test the molecular phenotype of these strains. Our current data support a role for fitness trade-offs driving divergence between contrasting intracellular and extracellular niches, highlighting nitrogen as a key environmental axis. Fitness trade-offs may, therefore, be a general, simple mechanism acting to reinforce symbiosis, contributing to evolutionary stability.