The objective of the present work was the synthesis of 1-[5-acetyl-4 (4-substituted phenyl)-2,6-dimethyl-1,4-dihydroxypyridine-3-yl]-ethan-1-one and evaluation of in vitro anticancer activity. Based on this a new series of compound had been planned to synthesize by reacting acetyl acetone with various aromatic aldehydes in the presence of ammonium acetate. The in vitro anticancer activities were carried out against Human small cell lung DMS 114 cell line and Human colon cancer cell line HCC 2998 and MTT assay was used to analyze the cell growth inhibition of the both. The results had been displayed that compound B1-B4 were possessed an excellent anticancer activity (at 25 µg/ml) against both Human small cell lung DMS 114 cancer cell line and Human colon cancer cell line HCC 2998 and doxorubicin (at 10µg/ml) was used as a standard drug for Human small cell lung DMS 114 cancer cell line and 5-Fluro uracil (5-FU) for Human colon cancer cell line HCC 2998. In the present study IC50  values below 4 µg/ml were displayed by the  compound B1 (IC50 of 4.0 µg/ml), B2 (IC50 of 3.4 µg/ml), B3 (IC50 of 3.2 µg/ml) and  B4 (IC50 of 3.1 µg/ml) against Human  small cell lung DMS 114 cancer cell line and  B1( IC50 of 3.5 µg/ml), B2 (IC50 of 3.2 µg/ml), B3 (IC50 of 2.9 µg/ml) and B4 (IC50 of 2.9 µg/ml) against Human colon cancer cell line HCC 2998. The IC50 values of standard drugs doxorubicin and 5-FU were found to be 1. 2 µg/ml and 1.1 µg/ml.