This paper describes the reversible activation of a mechanosensitive channel via a light-sensitive lipid mimic. For these experiments, the mechanosensitive channel of large conductance (MscL) protein from Lactococcus lactis and Escherichia coli was reconstituted in lipid bilayers composed of 80 mol% 1,2-dioleoyl-sn-glycero-3-phosphocholine and 20 mol% di-(5-{[4-(4-butylphenyl)azo]phenoxy}pentyl)phosphate (4-Azo-5P). Light-induced isomerization of the azobenzene moiety of 4-Azo-5P from trans to cis was used to activate MscL.