Abstract: Estimation of the time since death (postmortem interval [PMI]) is one of the most difficult problems in forensic investigations, and many methods currently are utilized to estimate the PMI. The goal of this study was to investigate the changes of attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of rat brain from postmortem time 0–144 h. The intensity ratios of major absorbance bands were examined (I1066/I1392, I1168/I1392, I1234/I1454, I1301/I1392, I1647/I2956, I2921/I2850, and I1647/I1539). The spectra of rat brain displayed prominent changes with increasing PMI. The band at 2871 and 1737 per cm became weak with the time increasing and even disappeared at postmortem 96 and 72 h, respectively. A close linear correlation was shown between the relative absorption intensity and the PMI, and the I1234/I1454 offered a stronger correlation (r = 0.973). Our results indicate that ATR-FTIR spectroscopy may be a useful technique for estimating the PMI.