The underlying molecular mechanisms for anastomotic leakage (AL) after colorectal surgery are unknown and there are no therapeutics for AL prevention. Our aim was to correlate endogenous matrix metalloproteinase (MMP) activity, collagen concentration, and collagen/MMP/cytokine mRNA levels with anatomic location in human colorectal tissue. We enrolled 22 patients in this prospective study: 7 underwent elective laparoscopic sigmoid resection and 15 underwent low anterior resection for colorectal cancer. Full-thickness intestinal tissue rings from anastomoses constructed with a circular stapler were used for the determination of the MMP activity, tissue collagen concentration and mRNA levels. COL1A1 (p = 0.017) and COL3A1 (p = 0.0013) mRNA levels were lower in rectal tissue than in colonic samples. Neither MMP activities nor collagen concentrations differed significantly between the two anatomic locations. By elucidating the factors responsible for the decreased collagen production we may identify specific molecular targets in AL prophylaxis.