Fossil energy inputs and greenhouse gas (GHG) emissions associated with the cultivation and transport of sugarcane (Saccharum officinarum) for bioethanol production in Tanegashima, Japan, were estimated by life cycle assessment (LCA). The aim was to understand the effects of combined systems of polyethylene mulching treatment (mulching at planting and every ratooning, MM; mulching only at planting, MU; and untreated, i.e., no mulching at all, UU) and cultivar (a cold-tolerant genotype, NiTn18, and a conventional variety, NiF8). The mulch treatments and cultivars were combined to create six cultivation systems that were used to conduct a comparative assessment of cradle-to-gate energy inputs and emissions for bioethanol production. The LCA results showed that the energy inputs and GHG emissions resulting from the MM/NiF8 system were 6.29 MJ L−1 and 0.500 kg CO2e L−1, which were 14% and 23% lower, respectively, than the corresponding values in the UU/NiF8 system. In contrast, the MU/NiF8 system increased the environmental loads slightly. The use of NiTn18 improved sugarcane performance and ethanol yields substantially as compared with NiF8, reducing energy inputs to 5.38, 5.24, and 5.55 MJ L−1 and GHG emissions to 0.473, 0.450, and 0.441 kg CO2e L−1 for the UU, MU, and MM treatments, respectively. The energy inputs and GHG emissions were similar among the systems, indicating that more flexible mulching treatments might be acceptable in the NiTn18 systems than in the NiF8 systems. The energy inputs and GHG emissions resulting from the UU/NiTn18 system were 14% and 5% lower, respectively, than those of the MM/NiF8 system, suggesting that it may be possible to overcome the handicap of sugarcane production in cold conditions by breeding cold-tolerant cultivars.