Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly-constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A (CNA) as a target protein candidate. R5G42 binds CNA in vitro and stimulates its phosphatase activity. When expressed transiently in human cells, R5G42 induces the dephosphorylation of Bad. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries in order to interrogate the proteome and chart molecular regulatory networks.