Persistent inward currents (PICs) are widely reported in rodent spinal neurons. A distinctive pattern observed recently is staircase-like PICs induced by voltage ramp in serotonergic neurons of mouse medulla. The mechanism underlying this pattern of PICs is unclear. Combining electrophysiological, pharmacological, and computational approaches, we investigated the staircase PICs in serotonergic neurons of medulla in ePet-EYFP transgenic mice (postnatal days 1-7). Staircase PICs induced by 10-s voltage biramps were observed in 70% of serotonergic neurons ( = 73). Staircase PICs activated at -48.8 ± 5 mV and consisted of two components, with the first PIC of 45.8 ± 51 pA and the second PIC of 197.3 ± 126 pA ( = 51). Staircase PICs were also composed of low-voltage-activated sodium PIC (Na-PIC; onset -46.2 ± 5 mV, = 34), high-voltage-activated calcium PIC (Ca-PIC; onset -29.3 ± 6 mV, = 23), and high-voltage-activated tetrodotoxin (TTX)- and dihydropyridine-resistant sodium PIC (TDR-PIC; onset -16.8 ± 4 mV, = 28). Serotonergic neurons expressing Na-PIC, Ca-PIC, and TDR-PIC were evenly distributed in medulla. Bath application of 1-2 μM TTX blocked the first PIC and decreased the second PIC by 36% ( = 23, < 0.05). Nimodipine (25 μM) reduced the second PIC by 38% ( = 34, < 0.001) without altering the first PIC. TTX and nimodipine removed the first PIC and reduced the second PIC by 59% ( = 28, < 0.01). A modeling study mimicked the staircase PICs and verified experimental conclusions that sequential activation of Na-PIC, Ca-PIC, and TDR-PIC in order of voltage thresholds induced staircase PICs in serotonergic neurons. Further experimental results suggested that the multiple components of staircase PICs play functional roles in regulating excitability of serotonergic neurons in medulla. Staircase persistent inward currents (PICs) are mediated by activation of L-type calcium channels in dendrites of mouse spinal motoneurons. A novel mechanism is explored in this study. Here we report that the staircase PICs are mediated by sequentially activating sodium and calcium PICs in serotonergic neurons of mouse medulla.