The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P<0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10mM SrCl2+5μgmL-1 cytochalasin B for 5-6h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P>0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P>0.05). Oocytes from juvenile mice activated in 10mM SrCl2 for 2h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P<0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.