Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage