Cultured rat hippocampal network on a multi electrode array (MEA) dish is a useful model for analyzing network electrical dynamics and its developmental changes. Neurons autonomously form a complex network on a MEA dish and spontaneous electrical activity is often observed without any input from the external world. The spontaneous activity is generated by synaptic interactions between neurons and reflects an internal biochemical state of a whole neuronal network. The origin of electrical activity is electrochemical potential generated by active transport of ions, which requires catabolism of ATP. We elucidated the relationship between spontaneous electrical activity and external glucose concentration. The spontaneous activity changed transiently, depending on glucose concentrations. The number of electrical spikes in spontaneous activity increases depending on the concentration of external glucose concentration. Interestingly, this increase is not only suppressed but turn to decrease. In the case of glucose concentration is 17.56 mM, the number of activity is the most, and then it decreases in the case of glucose concentration is more than 20 mM. In addition, the decrease of neuronal activity at a high glucose concentration is not influenced by the blockade of inhibitory synaptic activity. These results suggest that a cultured neuronal network has optimal glucose concentration 17.56 mM, which corresponds to the concentration of glucose in a culture medium.