Anastomotic leakage (AL) is a devastating complication after colorectal surgery, possibly due to the loss of stabilizing collagen fibers in the submucosa. Our aim was to assess the formation of collagen in the colon versus the rectum with or without transforming growth factor (TGF)-β1 exposure in a human cellular model of colorectal repair. Primary fibroblasts were isolated by an explant procedure from clinically resected tissue rings during anastomosis construction in 19 consecutive colorectal patients who underwent laparoscopy. The cells, identified as fibroblasts by morphologic characteristics and flow cytometry analysis (CD90⁺), were cultured for 8 days and in 12 patients in the presence of 1 ng/mL TGF-β1. Total collagen deposition was measured colorimetrically after Sirius red staining of fixed cell layers, and type I, III, and VI collagen biosynthesis and degradation were specifically determined by the biomarkers PINP, PRO-C3, PRO-C6, and C3M in conditioned media by competitive enzyme-linked immunosorbent assays. Total collagen deposition by fibroblasts from the colon and rectum did not significantly differ. TGF-β1 treatment increased PINP, PRO-C6, and total collagen deposition. Mechanistically, TGF-β1 treatment increased COL1A1 and ACTA2 (encoding α-smooth muscle actin), and decreased COL6A1 and MMP2 mRNA levels in colorectal fibroblasts. In conclusion, we found no effect of anatomic localization on collagen production by fibroblasts derived from the large intestine. TGF-β1 represents a potential therapeutic agent for the prevention of AL by increasing type I collagen synthesis and collagen deposition.