Abstract Background Spider silk is a proteinaceous fiber with remarkable mechanical properties spun from spider silk proteins (spidroins). Engineering spidroins have been successfully produced in a variety of heterologous hosts and the most widely used expression system is Escherichia coli (E. coli). So far, recombinantly expressed spidroins often form insoluble inclusion bodies (IBs), which will often be dissolved under extremely harsh conditions in a traditional manner, e.g. either 8 mol/L urea or 6 mol/L guanidine hydrochloride, highly risking to poor recovery of bioactive proteins as well as unexpected precipitations during dialysis process. Results Here, we present a mild solubilization strategy—one-step heating method to solubilize spidroins from IBs, with combining spidroins’ high thermal stability with low concentration of urea. A 430-aa recombinant protein (designated as NM) derived from the minor ampullate spidroin of Araneus ventricosus was expressed in E. coli, and the recombinant proteins were mainly present in insoluble fraction as IBs. The isolated IBs were solubilized parallelly by both traditional urea-denatured method and one-step heating method, respectively. The solubilization efficiency of NM IBs in Tris-HCl pH 8.0 containing 4 mol/L urea by one-step heating method was already comparable to that of 7 mol/L urea with using traditional urea-denatured method. The effects of buffer, pH and temperature conditions on NM IBs solubilization of one-step heating method were evaluated, respectively, based on which the recommended conditions are: heating temperature 70–90 °C for 20 min, pH 7.0–10, urea concentration 2–4 mol/L in normal biological buffers. The recombinant NM generated via the one-step heating method held the potential functions with self-assembling into sphere nanoparticles with smooth morphology. Conclusions The one-step heating method introduced here efficiently solubilizes IBs under relatively mild conditions compared to the traditional ones, which might be important for the downstream applications; however, this protocol should be pursued carefully in terms of urea-induced modification sensitive applications. Further, this method can be applied under broad buffer, pH and temperature conditions, conferring the potential to apply to other thermal stable proteins.