The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines. Display omitted •501Y.V2 showed no higher infectivity in cells with hACE2 comparing to 614G variant•501Y.V2 showed increased infectivity in cells with mACE2 compared to 614G variant•501Y.V2 escaped neutralization by most of neutralizing monoclonal antibodies•501Y.V2 significantly compromised the inhibitory effects of polyclonal antibodies Experiments with pseudotyped viruses show that the 501Y.V2 variant of SARS-CoV-2 exhibits resistance to neutralization from monoclonal antibodies and sera from convalescent as well as immunized individuals, predominantly due to the E484K and N501Y mutations in the receptor-binding domain of the viral spike protein.