Epilepsy provides a unique opportunity to study human cellular electrophysiology through in vivo and in vitro approaches. In vivo recordings offer advantages in action potential pattern reconstruction and connection preservation, while in vitro studies allow broader tissue exploration within a confined space of cut connections, yet coherent investigation in these distinct conditions is lacking. This study compared single-neuron firing properties in four epileptic patients using identical 24-channel laminar microelectrodes with 150 μm inter-contact distance for both their in vivo and in vitro intracortical recordings. Automated spike sorting algorithms were employed for single-unit analysis, based on which unit waveforms, correlograms, firing rates, burstiness index, and determination of excitatory and inhibitory features were calculated. Of the 330 single neurons identified in vitro and 22 in vivo, in vitro cells exhibited significantly higher overall firing rates (2.73 Hz vs. 1.32 Hz in vivo). Principal cell firing rates were similar between conditions, while interneurons fired at a higher rate in vitro (2.93 vs. 0.74 Hz). Burstiness was significantly higher in vivo (6.986%) than in vitro (3.506%). Strong correlations were observed between patient-wise in vivo and in vitro unit numbers (r=0.703), as well as layer-specific analysis (0.926 for firing rates, 0.762 for burstiness indices). Discrepancies in firing rates and burstiness underscore differences between in vivo and in vitro conditions. In vitro neurons, particularly inhibitory cells with limited synaptic connections, exhibited higher activity than those in the intact neocortex. Lower burstiness in vitro may be attributed to altered environments and partial loss of synaptic inputs.