Background <p> Clinically, histological findings in skin injected with autologous dermal fibroblasts (DFs) are rare, and DF heterogeneity is neglected in autologous DF therapy. This study was designed to compare the histological findings in pig skin injected with autologous superficial DFs and deep DFs and to further explore the necessity of utilizing DF heterogeneity when autologous DF injection is performed.</p> Methods <p> Superficial and deep DFs were isolated and cultured from the dorsum of 3 female red Duroc pigs. Areas were injected with 1 ml (4×10⁷) of autologous superficial and deep DFs, or phosphate-buffered saline solution and served as the superficial group (n=3), the deep group (n=3), and the control group (n=3), respectively. Full-thickness skin samples were harvested one week later and subjected to hematoxylin &amp; eosin (H&amp;E) staining, Masson’s trichrome staining, and immunohistochemical staining for proliferating cell nuclear antigens and α-smooth muscle actin.</p> Results <p> H&amp;E staining revealed that several “cell clumps” were present in the subcutaneous fat of the superficial and deep groups but were absent in the control group. Masson’s trichrome staining dyed the “cell clumps” green to some extent in both the superficial group and the deep group. Immunohistochemical staining revealed that proliferating cell nuclear antigen expression was significantly higher in the superficial group than in the deep group, but α-smooth muscle actin expression was significantly lower in the superficial group than in the deep group.</p> Conclusions <p> The histological findings in pig skin injected with superficial/deep autologous DFs clearly differed, indicating that greater consideration should be given to DF heterogeneity after autologous DF injection.</p> Level of Evidence <p>Not gradable</p>