The aim of this study was to determine the effect of prepartum somatotropin injection in late-pregnant Holstein heifers on metabolism, milk production and resumption of postpartum ovulation. For this study, 31 late-pregnant Holstein heifers were used. The heifers were assigned randomly into two treatments: (1) 500 mg sc injections of somatotropin (somatotropin treatment, n = 15) at −35 and −21 days, and, if pertinent, at −7 days from expected calving date and (2) no treatment (control group, n = 16). Blood samples were collected weekly from −5 to 7 weeks after calving. Heifers with progesterone concentrations in plasma above 1 ng/ml in two consecutive postpartum samples were considered as having resumed ovarian activity. A higher proportion (P = 0.04) of heifers treated with somatotropin resumed ovarian activity in the first 7 weeks post partum (73.3%; 11/15) compared with the control group (37.5%; 6/16). A higher number (P = 0.02) of heifers in the somatotropin treatment group also ovulated during the first postpartum follicular wave (53.3%; 8/15) compared with the control group (12.5%; 2/16), as indicated by the number of heifers ovulating in the first 3 weeks post partum. Pregnancy rate was not affected by treatments (P > 0.10) and averaged 40.0% (6/15) in somatotropin-treated and 25.0% (4/16) in control heifers when evaluated up to 150 days in milk. Somatotropin treatment increased the average daily milk production by 2.8 kg/cow per day (P < 0.0001) and reduced the somatic cell count (P = 0.009). Plasma IGF-I was higher (P < 0.05) for somatotropin-treated heifers in the prepartum period. Insulin and body condition score were higher (P < 0.05) and non-esterified fatty acids were lower (P < 0.05) for somatotropin-treated cows in the early postpartum period. In conclusion, somatotropin injection during the prepartum period in late-pregnant Holstein heifers was able to increase the proportion of heifers resuming ovarian activity early post partum, inspite of higher milk production.