Cytoplasmic calcium (Ca2+) exerts a wide range of important physiological roles in excitable cells and glia. In neuronal pre-synaptic terminals nanomolar elevation of the cytoplasmic Ca2+ concentration [Ca2+]i generated by the voltage change during the action potential opens voltage-gated Ca2+ channels and confers supralinear scaling between [Ca2+]i and transmitter release. Action potentials also generate [Ca2+]i changes in the primary axon including the axon initial segment (AIS) and node of Ranvier but their precise source and role remain poorly understood. Here, taking advantage of the high sensitivity of one-photon epifluorescence imaging we investigated Ca2+ in somatosensory neocortical layer 5 axons filled with Ca2+ indicators during somatic patch-clamp recordings in acute brain slices from young-adult rats. Using the high-affinity indicator OGB-1 and correcting fractional fluorescence change (ΔF/F) to background fluorescence revealed that a brief subthreshold depolarization (5 EPSPs with a ~17 mV peak amplitude) generated a substantial and transient increase in [Ca2+]i at the AIS and nodes of Ranvier (ΔF/F 12.2 ± 1.2 % and 9.3 ± 0.9 %, respectively, n = 9) but not in the internodal domains (1.18 ± 0.35%; ANOVA with multiple comparisons, P = 0.0001 and P < 0.0001, n = 9). As expected, action potentials (~100 mV in peak amplitude) evoked larger [Ca2+]i elevations in the AIS and nodes, up to a ΔF/F of 35.9 ± 4.7% and 22.2 ± 3.2% (n = 6 axons) which subsequently spread into the internodes, soma and dendrites. Surprisingly, pharmacological experiments with a wide range of voltage-gated Ca2+ channel toxins and blockers revealed only a ~10% contribution during the AP-evoked [Ca2+]i transient in the AIS without an effect on the subthreshold-evoked [Ca2+]i (-3% with the T-type blocker TTA-P2 [1 µM], P > 0.42, n = 4). Based on > 8 compounds tested, the activity-dependent [Ca2+]i was only reduced with the voltage-gated Na+ channel blocker tetrodotoxin (TTX, 1.0 µM). TTX blocked both the subthreshold-depolarization evoked Ca2+ (91.5 ± 2.3% block, P = 0.0001, n = 4) and completely abolished AP-induced [Ca2+]i elevations (96 ± 1% block paired t-test, P < 0.0009, n = 4). Based on these data we hypothesized that Ca2+ enters the axon through the native voltage-gated Na+ channels, present at very high densities in the AIS (~400 channels µm-2). Such unconventional route has been reported in a few cell types and species, including the squid giant axon [1]. To test this we used a recently developed strategy combining a low-affinity indicator OGB-5N with one-photon imaging at the highest possible frame rate of 20 kHz [2] and estimated the time course of Ca2+ current based on optically recorded and temporally differentiated ΔF/F. Results from multiple experiments (n = 9 axons) suggest a microsecond precise alignment of the Ca2+ current with Na+ current (~200 µs activation time constant at room temperature at -35 mV). Taken together, we propose that in the neocortical pyramidal neuron axon initial segment native axonal Na+ channels are partially permeable to Ca2+, endowing Na+-channel enriched microdomains with a submillisecond rapid Ca2+ entry pathway and, in concert with amplification from intracellular organelles, enables the tuning of cytoplasmic Ca2+ to the fast axonal action potential dynamics.