The presence of perchlorate in the Martian soil may limit in-situ resource utilization (ISRU) technologies to support human outposts. In order to exploit the desiccation, radiation-tolerant cyanobacterium Chroococcidopsis in Biological Life Support Systems based on ISRU, we investigated the perchlorate tolerance of Chroococcidopsis sp. CCMEE 029 and its derivative CCMEE 029 P-MRS. This strain was obtained from dried cells mixed with Martian regolith simulant and exposed to Mars-like conditions during the BIOMEX space experiment. After a 55-day exposure of up to 200 mM perchlorate ions, a tolerance threshold value of 100 mM perchlorate ions was identified for both Chroococcidopsis strains. After 40-day incubation, a Mars-relevant perchlorate concentration of 2.4 mM perchlorate ions, provided as a 60 and 40% mixture of Mg- and Ca-perchlorate, had no negative effect on the growth rate of the two strains. A proof-of-concept experiment was conducted using Chroococcidopsis lysate in ISRU technologies to feed a heterotrophic bacterium, i.e. an Escherichia coli strain capable of metabolizing sucrose. The sucrose content was fivefold increased in Chroococcidopsis cells through air-drying and the yielded lysate successfully supported the bacterial growth. This suggested that Chroococcidopsis is a suitable candidate for ISRU technologies to support heterotrophic BLSS components in a Mars-relevant perchlorate environment that would prove challenging to many other cyanobacteria, allowing a ‘live off the land’ approach on Mars.