All ionoregulating marine fishes examined to date utilize seawater-type ionocytes expressing the apical Cl- channel, cystic fibrosis transmembrane conductance regulator (Cftr) to secrete Cl⁻. We performed transcriptomic, molecular, and functional studies to identify Cl⁻ transporters in the seawater-type ionocytes of sea lamprey (Petromyzon marinus). Gill cftr expression was minimal or undetectable in larvae and post-metamorphic juveniles. We identified other Cl⁻ transporters highly expressed in the gills and/or upregulated following metamorphosis and further investigated two candidates that stood out in our analysis, a Ca²⁺-activated Cl⁻ channel, anoctamin 1 (ano1), and the Clc chloride channel family member 2 (clcn2). Of these, ano1 was expressed 10-100 times more than clcn2 in the gills; moreover, ano1 was upregulated during seawater acclimation, while clcn2 was not. Using an antibody raised against sea lamprey Ano1, we did not detect Ano1 in the gills of larvae, found elevated levels in juveniles and observed a 4-fold increase in juveniles after seawater acclimation. Ano1 was localized to seawater-type branchial ionocytes but, surprisingly, was localized to the basolateral membrane. In vivo pharmacological inhibition experiments demonstrated that a DIDS-sensitive mechanism was critical to the maintenance of osmoregulatory homeostasis in seawater- but not freshwater-acclimated sea lamprey. Taken together, our results provide evidence of a Cftr-independent mechanism for branchial Cl⁻ secretion in sea lamprey that leverages Ano1-expressing ionocytes. Once further characterized, the Cftr-independent, Ano1-rich ionocytes of sea lamprey could reveal novel strategies for branchial Cl⁻ secretion, whether by Ano1 or some other Cl⁻ transporter, not previously known in ionoregulating marine organisms.