This study characterized the local effects of extracellular osmolality and prolactin (PRL) on branchial ionoregulatory function of a euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus). First, gill filaments were dissected from freshwater (FW)-acclimated tilapia and incubated in four different osmolalities, 280, 330, 380 and 450 mOsm/kg. The mRNA expression of Na⁺/K⁺-ATPase a1a (NKA a1a) and Na⁺/Cl^- cotransporter (NCC) showed higher expression with decreasing media osmolalities, while Na⁺/K⁺/2Cl^- cotransporter 1a (NKCC1a) and PRL receptor 2 (PRLR2) mRNA levels were upregulated by increases in media osmolality. We then incubated gill filaments in media containing ovine PRL (oPRL) and native tilapia PRLs (tPRL₁₇₇ and tPRL₁₈₈). oPRL and the two native tPRLs showed concentration-dependent effects on NCC, NKA a1a and PRLR1 expression; Na⁺/H⁺ exchanger 3 (NHE3) expression was increased by 24 h of incubation with tPRLs. Immunohistochemical observation showed that oPRL and both tPRLs maintained a high density of NCC- and NKA-immunoreactive ionocytes in cultured filaments. Furthermore, we found that tPRL₁₇₇ and tPRL₁₈₈ differentially induce expression of these ion transporters according to incubation time. Together, these results provide evidence that ionocytes of Mozambique tilapia may function as osmoreceptors as well as directly respond to PRL to modulate branchial ionoregulatory functions.