While p53 levels and mutations correlate well with tumorigenesis, relatively little is known about the role of p53 variations in environmental adaptation. Myospalax baileyi and Microtus oeconomus, are native rodents living at the Qinghai-Tibet plateau and well acclimatized to hypoxia, cold, and/or high CO2 environments. The CDS of p53 from M. baileyi and M. oeconomus were cloned and sequenced. Evolutionary analysis distinguished the codon 104 variation from other sites as an important mutation correlated with the adaptation of these two rodents to plateau environments. The S104N of M. baileyi p53 is different from two of its close species Myospalax cansus and Spalax judaei living at lowland. The S104E of M. oeconomus p53 shared with only four fishes and squid, most of which were reported to be hypoxia-tolerant. To address the functional characteristics of their p53, the p53-null human non-small lung cancer cells NCI-H1299 (ATCC CRL-5803) were transfected with expression plasmids of M. baileyi, M. oeconomus and human p53. As determined by dual-luciferase assays, while the S104N of M. baileyi p53 induced stronger transactivation of pro-apoptotic target genes IGFBP3 and Apaf1 (1.9 and 1.5 folds, respectively; p < 0.001), the S104E of M. oeconomus p53 was responsible for the deficient transactivation of IGFBP3, Apaf1 and bax (0.3, 0.4 and 0.5 folds, respectively; p < 0.001). Values are means ± SD, compared by one-way ANOVA (n = 3). Using site-directed mutagenesis and dual-luciferase assays, we demonstrated that the codon 104 variation contributes to its sensitivity to cold and acidic stresses. While M. baileyi wt p53 induced IGFBP3 transactivation at 37 °C is 1.5 folds higher than that at 30 °C (p < 0.001), both N104S and N104E mutants had similar transactivation to IGFBP3 between 30 °C and 37 °C (p > 0.05). At pH 6.0, IGFBP3 induced by M. baileyi wt p53 decreased by 40% compared with at pH 7.4 (p < 0.01), whereas IGFBP3 induced by N104E mutant only decreased by 10% (p < 0.05). We propose that these p53 codon 104 variations are an outcome of environmental adaptation and evolutionary selection that enhances cellular survival strategies befitting the hypoxic, cold, and high CO2 (for M. baileyi) environments at the Qinghai-Tibet plateau.