Abstract: Objective To investigate the differences in serum biochemistry between specific pathogen-flee (SPF) and conventional aged Wistar rats. Methods Coulter-JT Analyzer was used to measure the values of serum biochemistry in the two grades of rats. Results The serum levels of alanine aminotransferase (ALT), total protein (TP), alkaline phosphatase (ALP), total cholesterol (TC), triglyceride (TG), blood urea nitrogen (BUN), creatinine, Fe, P, glucose, uric acid (UA), and low density lipoprotein (LDL) were very significantly different between male and female Wistar rats of either conventional or SPF grade (P<0.01), which also had significant difference in albumin, lactate dehydrogenase (LDH) and apolipoprotein B (ApoB) (P<0.05). Between male aged Wistar rats of the two grades, the differences of TP, albumin, albumin/globulin (A/G) ratio, TC, TG, blood glucose, ApoA1, ApoB, UA, high-density lipoprotein (HDL), LDL, and glutamic oxalacetic transaminase (GOT) were very significant (P<0.01), with also significant differences in ALT, Fe, Mg (P<0.05). Between the female rats of the two grades, the serum levels of ALT, TP, albumin, A/G ratio, ALP, TG, BUN, creatinine, Fe, ApoA1, APOB, HDL, LDL, and bile acids were very significantly different (P<0.01), and Mg was significantly different (P<0.05). Conclusion Different microbiological profiles affect serum biochemistry of aged Wistar rats.