Water-soluble polyisoprene polyrotaxanes based on cyclodextrins (CDs) were synthesized via rotaxa-polymerization for the first time in a single step in one pot. Hydrophilic beta-CD derivatives were utilized to complex isoprene, which was copolymerized with bulky, hydrophilic comonomers that acted as stoppers. The effect of the CD derivatives, stoppers, initiators and monomer content on the yield, CD coverage, and solubility of the polymeric product were investigated. The best yield of water-soluble polymer was obtained for the stopper comonomer that consisted of poly(ethylene glycol) methyl ether methacrylate (PEG MA). The highest CD content (67 wt %) on the polyisoprene backbone was observed for randomly methylated beta-CD (RAMEB) with a 1:1 molar ratio of isoprene to CD in the feed and for the thermal radical initiator VA-044. Excess isoprene over CD increases the yield of polyrotaxane but lowers its CD content and gives rise to partially insoluble byproducts. Polymer backbones (30 kDa) with high molar masses were achieved.