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We examined the efficacy of tenofovir disoproxil fumarate (TDF) in blocking simian human immunodeficiency virus (SHIV) infection in Chinese rhesus macaques. Once weekly for 14 weeks or until a macaque became infected, 12 male macaques were inoculated intrarectally with amounts of SHIVSF162P3 (10 median tissue culture infective doses; 3.8×105 virus particles) that were ∽5-fold higher than the human immunodeficiency virus type 1 RNA levels noted in human semen during an acute infection. Of the 12 macaques, 4 received oral TDF daily, 4 received oral TDF once weekly, and 4 (control animals) received no TDF. The control animals became infected after receiving a median of 1.5 virus inoculations; macaques receiving TDF daily (1 macaque remained uninfected after 14 inoculations) and those receiving TDF weekly became infected after a median duration of 6.0 and 7.0 weeks, respectively. Although infection was delayed in treated macaques, compared with control macaques, the differences were not statistically significant (P=.315); however, the study was limited by the small numbers of animals evaluated and the variability in blood levels of TDF that resulted from oral dosing. These data demonstrate that treatment with oral TDF provided partial protection against SHIV infection but ultimately did not protect all TDF treated animals against multiple virus challenges.