Water-soluble linear polymers Gu(m)BA(n) (m/n = 18/6, 12/12, and 6/18) with multiple guanidinium ion (Gu(+)) and boronic acid (BA) pendants in their side chains were synthesized as ATP-responsive modulators for enzyme activity. Gu(m)BA(n) polymers strongly bind to the phosphate ion (PO4-) and 1,2-diol units of ATP via the Gu(+) and BA pendants, respectively. As only the Gu(+) pendants can be used for proteins, Gu(m)BA(n) is able to modulate the activity of enzymes in response to ATP. As a proof-of-concept study, we demonstrated that trypsin (Trp) can be deactivated by hybridization with Gu(m)BA(n). However, upon addition of ATP, Trp was liberated to retrieve its hydrolytic activity due to a higher preference of Gu(m)BA(n) toward ATP than Trp. This event occurred in a much lower range of [ATP] than reported examples. Under cellular conditions, the hydrolytic activity of Trp was likewise modulated.