Background <p>Intracellular calcium (Ca²⁺) signaling is essential for oocyte maturation, activation, and fertilization, with repetitive Ca²⁺ transients elicited at fertilization being critical for egg activation and early embryonic development. Mouse oocytes express several non-selective cation channels to support these oscillations, including TRPV3, a member of the transient receptor potential (TRP) channel family. In addition to Ca²⁺, TRPV3 mediates zinc (Zn²⁺) influx, which is a modulator of cortical granules (CGs) distribution and actin organization. In mammals, CG exocytosis mediates the fertilization-induced block to polyspermy, and pharmacological activation of TRPV3 in mouse oocytes elicits Ca²⁺ influx sufficient to trigger activation and parthenogenesis. Despite these critical roles in mice, the expression and function of TRPV3 in other mammals remain unexplored. Here, we evaluate the functional expression of TRPV3 channels in cat oocytes.</p> Methods <p>Ovaries from domestic cats were obtained during ovariohysterectomies, and oocytes were isolated and matured <i>in vitro</i>. <i>Trpv3</i> expression was assessed by RT-PCR from ovaries and germinal vesicle (GV) and metaphase II (MII) oocytes, while TRPV3 localization was evaluated by immunocytochemistry. Mouse WT and TRPV3-knockout oocytes were used as controls for antibody specificity. Functional channel activity was examined using Ca²⁺ imaging following addition of the TRPV3 agonist 2-APB. Three-dimensional modelling, molecular docking, and comparative sequence analysis of feline, mouse, and human TRPV3 proteins were aligned with MAFFT, focusing on identical and biochemically similar residues within the 2-APB-binding sites as well as pore-forming and temperature-sensing domains to assess potential species-specific functional differences.</p> Results <p>We detected <i>Trpv3</i> transcripts in cat ovaries and in GV and MII oocytes. Immunocytochemistry confirmed TRPV3 protein localization at the oocyte membrane in cats, consistent with reports in mouse oocytes. Additionally, addition of 2-APB elicited robust intracellular increase in Ca²⁺ in MII cat eggs, demonstrating functional TRPV3 channel activity. Comparative analyses revealed non-conservative substitutions in feline TRPV3 compared to mouse and human TRPV3, particularly within the pore-forming, channel gating, and temperature sensing regions, offering a molecular explanation for species-specific differences in TRPV3 function.</p> Conclusions <p>Our results demonstrate functional TRPV3 expression in domestic cat oocytes. We find distinctive features in feline TRPV3 compared to rodent and human orthologs. These insights support the development of tailored artificial oocyte activation protocols in cats, with potential applications to Assisted Reproductive Technologies (ART) for endangered felids.</p>