We investigated the effect of chronic hypoinsulinemia on the level of synaptic activity and short-term plasticity in cultured hippocampal neurons. Hypoinsulinemia was induced by culturing mature (16-20 days in vitro) rat’s hippocampal neurons without insulin for 1, 2, and 4 days. The control insulin concentration was 100 nM. Spontaneous and evoked glutamatergic excitatory postsynaptic currents (sEPSC and eEPSC, respectively) in these neurons were analyzed using the whole-cell patch-clamp method and the method of local electrical stimulation of individual axon. Hypoinsulinemia during the 1st, 2nd and 4th days led to significantly reduction of the mean sEPSC’s frequency to 49.9 ± 15.8% (n = 6), 8.5 ± 7.7% (n = 6) and 16.6 ± 5.2% (n = 8) respectively, relative to control. Also, there was a decrease of the average sEPSC’s amplitudes to 52.6 ± 5.5% (n = 6), 36.6 ± 5.8% (n = 6) and 43.9 ± 8.4% (n = 8), respectively, relative to control. Quantal analysis of the sEPSC’s amplitudes showed a decrease of multivesicular glutamate release at the synapses under such conditions. Hypoinsulinemia caused a shift in the direction of short-term plasticity in glutamatergic hippocampal synapses from potentiation to depression. The paired-pulse ratio decreased from 1.83 ± 0.25 in the control to 0.59 ± 0.07, 0.77 ± 0.07, and 0.80 ± 0.06 after the 1st, 2nd, and 4th days under cultivation without insulin. Accordingly, the ratio of the coefficients of variation of eEPSC’s amplitudes (CV2/ CV1) increased from 0.82 ± 0.07 to 1.30 ± 0.28, 1.52 ± 0.27, and 1.61 ± 0.24. The presented results indicate a significant reduction of synaptic activity and decrease in the probability of multivesicular release of glutamate at the synapses of cultured hippocampal neurons under hypoinsulinemia.