This report describes in situ ion microprobe U–Pb dating of a protoconodont, an early Cambrian phosphate microfossil, using laterally high-resolution secondary ion mass spectrometry (NanoSIMS). On a single fragment of a fossil (approximately 850 μm × 250 μm) derived from a sedimentary layer in the Meishucunian Yuhucun Formation, Yunnan Province, southern China, 23 spots provide a 238U/206Pb isochron age of 547 ± 43 Ma (2σ, MSWD = 1.9), which is consistent with the depositional age, 536.5 ± 2.5 Ma estimated using zircon U–Pb dating of interbedded tuffs. However, five spots on a small region (approximately 250 μm × 100 μm) in the same protoconodont yield an isochron age of 417 ± 74 Ma (2σ, MSWD = 0.31), apparently younger than the formation age. The younger age might be attributable to a later hydrothermal event, perhaps associated with Caledonian orogenic activity recorded in younger zircon with an age of 420–440 Ma. We also measured 87Sr/86Sr ratios of the protoconodont by NanoSIMS. In the older domain, 19 spots give the 87Sr/86Sr ratio of 0.71032 ± 0.00023 (2σ), although seven spots on the younger region provide the ratio of 0.70862 ± 0.00045; this is significantly less radiogenic than the older domain. This is the first report of U–Pb age and Sr isotope heterogeneity within a single fragment of micro-fossil (215).