In this study, the expression and implication of acid-sensing ion channels 2 and 4 (ASIC2 and ASIC4) in the gonadal sex differentiation of Dicentrarchus labrax (D. labrax), subjected to increasing water temperatures during gonadal development, were evaluated. Two groups were selected: a control group (CG), in which the average water temperature was maintained at 15 °C and increased to 20 °C in 20 days until weaning; and an experimental group (EG), in which the water temperature was retained at 15 °C for 60 days; thereafter, the temperature was increased daily by 0.5 °C until it reached 20 °C up to the weaning time. Ten fish from the CG and 13 fish from the EG were sampled randomly on the 335th day after hatching (dph). A higher percentage of gonad differentiation in ovaries rather than in testes was observed in the EG compared to the CG (p = 0.01). ASIC2 and ASIC4 were detected for the first time in D. labrax ovaries by indirect immunofluorescence. Both ASIC2 and ASIC4 were expressed in previtellogenic oocytes of ovaries and in scattered cells within some testes, and were most likely intratesticular previtellogenic oocytes in both the CG and EG groups. The CG group showed a higher expression of ASIC4 than the EG cohort (p < 0.05). The results gathered in this study revealed the capacity of water temperature to influence both gonadal differentiation and growth in this gonochoristic fish species, and suggests the possible role of ASIC2 and ASIC4 in gonad differentiation and gamete development in D. labrax.