The influence of nutrient limitation on polyhydroxyalkanoate (PHA) accumulation was studied using a novel PHA-producing strain of Enterobacter sp. The effect of N/C ratio on growth and accumulation of PHA was studied by varying the ratio from 0.02 to 0.1. Biomass concentration, dry cell weight, protein content and the amount of PHA accumulated were estimated for each N/C ratio. It was found that the increase in N/C ratio resulted in increase in culture concentration up to 3.25 g/l of dry cell weight. Polyhydroxyalkanoate concentration was found to be maximum at N/C ratio of 0.04 (67.8 µg/ml), and further increase in N/C ratio resulted in lesser amount of PHA. Analytical procedures such as FTIR and NMR were done to validate the obtained PHA biopolymer.