Backgrounds: Canine mammary gland tumors (CMGTs) are heterogeneous tumors and share many similar features with human breast cancer. Despite the improvement of current treatment options, new treatment modalities are required to effectively kill tumor cells without general toxicity in the treatment of CMGTs. Photodynamic therapy (PDT) is a promising method for cancer treatment. However, there is a limited study evaluating the therapeutic efficacy of PDT in the treatment of CMGTs. Methods: In this context, we, for the first time, investigated the therapeutic potential of 5-aminolaevulinic acid (5-ALA) mediated PDT at 6 and 12 J/cm2 in two different subtypes [Tubulopapillary carcinoma (TPC) and carcinosarcoma (CS)] cells via different molecular analysis. The cytotoxic effects of 5-ALA/PDT on these cells were analyzed by intracellular PpIX level, WST-1 and ROS analysis. Furthermore, the underlying moleculer mechanism of 5-ALA/PDT mediated apoptotic effects on TPC and CS cells were evaluated Annexin V, AO/PI, RT-PCR and western blot analysis. Results: The 5-ALA/PDT treatment upon irradiation considerably inhibited the viability of both TPC and CS cells (p<0.01) and caused apoptotic death through elevated ROS levels, the activation of Caspase-9, and Caspase-3, and the overexpression of Bax. However, the response of TPC and CS cells to 5-ALA/PDT was different. Conclusions: Our preliminary in vitro findings provide novel insights into the molecular mechanisms underlying 5-ALA/PDT mediated apoptosis in both TPC and CS cells. However, the therapeutic response of CMGT cells to 5-ALA/PDT is limited. Keywords: 5-aminolaevulinic acid; Apoptosis; Canine mammary gland tumor; Carcinoma; Carcinosarcoma; Photodynamic therapy.