Prolactin (PRL) cells within the rostral pars distalis (RPD) of the euryhaline teleost tilapia, Oreochromis mossambicus, rapidly respond to a hyposmotic stimulus by releasing two distinct PRL isoforms, PRL188 and PRL177. Here, we describe how environmentally relevant temperatures affect the release and mRNA levels of PRL188 and PRL177 from RPDs and dispersed PRL cells. When applied under isosmotic conditions (330 mOsm/kg), a 6 °C rise in temperature stimulated the release of PRL188 and PRL177 from both RPDs and dispersed PRL cells under perifusion. When exposed to this same change in temperature, ~50% of dispersed PRL cells gradually increased in volume by ~8%, a response partially inhibited by the water channel blocker, HgCl2. Following their response to increased temperature, PRL cells remained responsive to a hyposmotic stimulus (280 mOsm/kg). The mRNA expression of transient potential vanilloid 4, a Ca2+-channel involved in hyposomotically-induced PRL release, was elevated in response to a rise in temperature in dispersed PRL cells and RPDs at 6 and 24 h, respectively; prl188 and prl177 mRNAs were unaffected. Our findings indicate that thermosensitive PRL release is mediated, at least partially, through a cell-volume dependent pathway similar to how osmoreceptive PRL release is achieved.