Spinach (Spinacia oleracea L.) is an important vegetable crop of which dioecy in nature has made cultivar improvement difficult using traditional breeding methods; therefore, production of high amount of disease free spinach is critical. To achieve the best explants and media for spinach tissue culture, the effects of two different plant growth regulators, two explants and cultivars on adventitious shoot regeneration were tested. The Analysis of Variance (ANOVA) showed that the effects of plant growth regulators on spinach tissue culture were significant; moreover, the effects of explants were not significant except on the regeneration phase. The best medium for callous induction was MS media containing 1.5 mgl-1 IAA + 2.5 mgl-1 GA3. The best medium for shoot regeneration was MS media contained 0.5 mgl-1 NAA + 2 mgl-1 GA3. The best rooting medium was MS medium containing 0.5 mgl-1 IBA. Results presented inhibitory effect of GA3 for callus and root formation; whereas, promote shoot development.