The PI3K/Akt/PTEN axis is one of the most important signaling pathways in tumorigenesis. Recently, mutation of PIK3CA has been highlighted due to the similarities of mutational hotspots in both dogs and humans. PIK3CA H1047R (c.3140A > G) has been discovered as the most common mutational hot spot in canine mammary tumor in recent studies, while the feature of PIK3CA-mutated canine mammary tumor is obscure. Methods: A total of 83 mammary samples classified as normal (n = 13), adenoma (n = 25), low-grade carcinoma (n = 21), and high-grade carcinoma (n = 24) were included in this study. Genomic DNA from each sample was extracted, amplified by conventional PCR, and analyzed through Sanger sequencing. Analysis for the expression of PIK3CA, Akt, p-Akt, and PTEN was performed by immunohistochemistry, and of Akt2 by RNA in situ hybridization. Results: PIK3CA H1047R mutation was detected in 14.3% (10/70) of tumor samples. Dysregulation of p-Akt, Akt2, and PTEN was observed in mammary tumor samples, but only PTEN dysregulation was associated with PIK3CA H1047R mutation. Conclusions: The present study showed that dysregulation of components in the PI3K/Akt/PTEN pathway is a feature of canine mammary tumors, but this dysregulation is not directly correlated to the PIK3CA H1047R mutation except for PTEN expression.