Bio-based thermoplastic natural rubber (TPNR) has recently received much attention due to its sustainability. TPNR based on natural rubber (NR), poly(lactic acid) (PLA), thermoplastic starch (TPS), and nano-precipitated calcium carbonate (NPCC) was fabricated using a twin-screw extruder with two different mixing sequences: MI (NPCC was first compounded with PLA) and MII (NPCC was initially compounded with TPS), and then converted to a sheet through cast sheet extrusion. A constant weight ratio of NR:PLA:TPS at 30:40:30 and varying concentrations of NPCC at 0.5, 1, 3, and 5 wt% were employed. The effects of NPCC and mixing sequence on the properties of NR/PLA/TPS/NPCC nanocomposites were investigated. The NR and TPS phases were dispersed in the PLA matrix. The nanocomposites loaded with a small amount of NPCC (0.5 and 1 wt%) showed increased tensile strength and Young's modulus. NPCC enhanced melt flowability, slightly improved the water vapor barrier property of the NR/PLA/TPS blend and caused decreased T-g, T-cc, and T-m of PLA in the nanocomposites. The PLA phase of the MI nanocomposites contained a higher amount of NPCC, consequently having greater PLA chain scission and poorer tensile properties than that of the MII nanocomposites.