This study is focused on optimizing the incubation conditions for c9, tl 1 CLA conjugated linoleic acid production by Laaobacillus acidophilus F0221 in MRSC broth (MRS broth supplemented with 0.5g·L-1 L-cysteine) and evaluating the effect of possible intestinal nutrients (non-digestible carbohydrates, monosaccharides and short chain fatty acids) on c9, t11 CLA production.L.acidophilus F0221 was grown in the MRSC broth supplemented with different concentrations (0, 0.1, 0.25, 0.5, 0.75.1.0, 1.5 and 2.0 mg·mL-1) of free linoleic acid and incubation was at different initial pH levels (5.5, 6.0, 6.5, 7.0, 7.5and 8.0) , temperatures (20, 25, 30, 37, 40, 45 and 50℃) and atmospheric conditions (aerobic and anaerobic conditions).The strain initially inoculated into the MRSC broth containing 0.5 g·L-1 linoleic acid at pH 6.5 followed for anaerobic incubation at 37℃ for 40h was found to be the most effective in promoting c9, t11 CLA production (105.43 μg·mL-1).Five oligosaccharides (xylooligosaccharide, fructooligosaccharide, isomaltooligosaccharide, inulin and galactooligosaccharide) , two polysaccharides (arabinogalactan and indigestible dextrin) and five monosaccharides (glucose, fructose, lactose, galactose and xylose) as the sole carbon source were added to the LA-MRSC (linoleic acid-MRSC) broth and incubated under these optimal conditions.Galactosaccharide, arabinogalactan, galactose and glucose had a higher growth promoting effect on c9, t11 CLA production (96.19-123.89μg·mL-1) than other carbon sources (22.62-75.92μg·mL-1).The presence of different short chain fatty acids (acetic acid, butyric acid, lactic acid and propionic acid) in the LA-MRSC broth, maximum c9, t11 CLA production (130.98μg·mL-1) was observed when acetic acid was used as a supplement.These results provide detailed parameters for the production of c9, t11 CLA by L.acidophilus F0221 and are valuable for further understanding of c9, t1 1 CLA formation in the human intestine by this strain.