Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, antibacterial,and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against humanliver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, thepresent study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After theexposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)(MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. Theresults showed a concentration-dependent significant reduction in the percentage cell viability and an alteration inthe cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56%by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrationswere found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT andNRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, andappeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resultedin significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer(A-549) cell lines.