The neurohypophysial nonapeptide arginine vasotocin (AVT) plays a role in regulation of osmotic balance in teleost fishes, but its mechanisms of action are not fully understood. Recently, is was discovered that nonapeptide receptors differentiated into V1a-type, several V2-type, and two isotocin (IT) receptor paralogs in teleost fishes, and it remains unclear which of these nonapeptide receptors mediate AVT’s action on gill osmoregulation. Here, we examine the role of nonapeptide receptors in gill osmoregulation by examining the transcriptional responses of gill nonapeptide receptors in euryhaline Amargosa pupfish (Cyprinodon nevadensis) acclimated to 7.5 ppt salinity, and then transferred to fresh water (0.3 ppt), seawater (35 ppt), or hypersaline (55 ppt) conditions. Transcript abundance of the teleost V1a-type receptor v1a2 was up-regulated over 4-fold in gill 24 h after transfer of fish from 7.5 ppt to 35 ppt or 55 ppt, and down-regulated upon transfer to 0.3 ppt, but returned to baseline levels by 14 d after transfer when fish had re-established osmolality. Transcripts for the nonapeptide degradation enzyme leucyl-cystinyl aminopeptidase (LNPEP) also increased in the gill of fish acclimating to 35 ppt, but returned to baseline levels by 14 d. To test whether AVT’s effects on the gill might be mediated by a V1a-type receptor, we also administered exogenous AVT or a V1-type receptor antagonist (Manning compound) to pupfish acclimated to 7.5 ppt prior to transfer of fish to 0.4 ppt or 35 ppt. AVT inhibited the increase in gill Na+/Cl- cotransporter 2 (ncc2) mRNA abundance that occurs when fish are transferred to hypo-osmotic environments, and V1-type receptor antagonism increased mRNA levels for ncc2 even without a change in salinity. Pupfish transferred to 35 ppt also exhibited elevated gill mRNA abundance for cystic fibrosis transmembrane conductance regulator (cftr), which diminished under V1-receptor inhibition. Taken together, these findings provide evidence for AVT acting via a V1-type receptor to regulate gill Cl- transport by inhibiting Cl- uptake and facilitating Cl- secretion during seawater acclimation.