Mild traumatic brain injury (mTBI) accounts for 80% of all TBI, may be associated with chronic impairments, and is difficult to diagnose due to a lack of objective markers. In this study, we investigated whether neurosteroids can serve as blood biomarkers for mTBI. Two cohorts of C57BL/6 mice were subjected to a model of mTBI combining impact with rotational acceleration or sham surgery. The first cohort underwent neurological testing for anxiety, balance, and locomotion before and after mTBI. For the second cohort, brains and plasma were collected 6 or 24 h after mTBI to measure steroid and neurosteroid levels by gas chromatography-tandem mass spectrometry. Traumatized mice exhibited significantly prolonged wake-up time from anesthesia, transiently increased beam-walk time, and mild astrogliosis compared with their control counterparts, but did not suffer from skull fractures, intracranial hemorrhage, or mortality. Isopregnanolone and 3β,5α-tetrahydrodeoxycorticosterone (ISODOC) were significantly decreased by more than 50% in brain parenchyma at 6 and 24 h after mTBI, while ISODOC was also significantly decreased in plasma (-75%). Therefore, ISODOC may be a candidate diagnostic biomarker for mTBI.