Unlike other domestic animals, in vitro maturation (IVM) of canine oocytes still has limited success. The present study investigated the effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. The donor bitches were categorized into three groups based on stage of estrus cycle: follicular (proestrus or estrous), luteal (diestrus) and anestrus. One ovary of each pair collected from 39 mature bitches was transported in Phosphate Buffer Saline (PBS) at 4 ?C while the other was transported at 37 ?C. A total of 1138 Grade I COCs obtained from all ovaries were grouped and matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH), essential and non-essential amino acids at 38.5 ?C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. The nuclear maturation rates were evaluated by aceto–orcein staining. Oocytes harvested from follicular and luteal ovaries have a significantly higher maturation rates (MI + MII) than the oocytes from anestrual ovaries in the 37 ?C group (p < 0.05). However, oocytes harvested fromanestrual ovaries transported at 4 ?C had the highest maturation (MI + MII) rate, and the difference between anestrual and luteal ovary groups was significant (p < 0.05). The oocytes from anestrual ovaries transported at 4 ?C have significantly higher maturation rates than those transported at 37 ?C (p < 0.0001). However, the transport temperature (37 or 4 ?C) did not significantly affect the maturation (MI + MII) rates of oocytes harvested from the luteal (p = 0.61) and follicular (p = 0.48) stage ovaries. It can be concluded from this study that (1) both transport temperature and transport temperature estrus cycle stage interaction effected the maturation rates, while estrus cycle stage alone did not, and (2) transporting canine ovaries at 4 ?C can improve in vitro maturation rates in oocytes harvested from anestrous ovaries.