We found better liver graft regeneration with hypothermic machine perfusion (HMP) compared with static cold storage (SCS) for the first time in our pilot study, but the underlying mechanisms are unknown. Upregulated heme oxygenase- (HO-) 1 expression has been reported to play a pivotal role in promoting hepatocyte proliferation. Here, we evaluated the novel role of HO-1 in liver graft protection by HMP. Rats with a heterozygous knockout of HO-1 (HO-1+/−) were generated and subjected to 3 h of SCS or HMP pre-half-size liver transplantation (HSLT) in vivo or 6 h of SCS or HMP in vitro; control rats were subjected to the same conditions (HO-1+/+). We found that HSLT induced significant elevation of the HO-1 protein level in the regenerated liver and that HO-1 haplodeficiency resulted in decreased proliferation post-HSLT. Compared with SCS, HMP induced significant elevation of the HO-1 protein level along with better liver recovery, both of which were reduced by HO-1 haplodeficiency. HO-1 haplodeficiency-induced decreased proliferation was responsible for the attenuated regenerative ability of HMP. Mechanistically, HO-1 haploinsufficiency resulted in suppression of hepatocyte growth factor (HGF)/Akt activity. Our results suggest that inhibition of HO-1 mitigates HMP-induced liver recovery effects related to proliferation, in part, by downregulating the HGF-Akt axis.