Fifty-two multiparous Brahman type cows with reproductive tract scoring (RTS) ≥4 at 45 days post-partum were randomly assigned to two groups of 26 cows each separated into an ad libitum suckling group (C) and treatment group (T). Calves in the T group were separated for 12 h during the night from 45 days post-partum to the onset of the breeding season. Body condition score (BCS) and body weight (BW) were recorded 45 days post-partum, at the start of the breeding season, and at pregnancy diagnosis. Calves were weighed at calving and weaning. Weaning weights were corrected to 205 days. BW and BCS at the onset of the breeding season were similar (p > 0.05) between the experimental groups. Calving to breeding intervals were 93 ± 18 d and 99 ± 22 d for T and C groups, respectively. Calving to conception intervals differed significantly between the groups (111 ± 10 d for T and 133 ± 19 d for C) and a similar result was obtained for the breeding to conception intervals (18 ± 15 d for T and 31 ± 19 d for C). Conception rates were 80% for the T group and 59% for the C group, which correlated better with BW than BCS at the onset of the breeding season. Weaning weights differed (p < 0.05) between C and T groups. From 45 days post-partum to the onset of the breeding season, cows in the T group experienced a positive energy balance (3%) while those in the C group had a negative energy balance (-0.1%). It was concluded that 12 h calf separation at night increases the conception rates and improves the calf weaning weights of Bos indicus beef cattle under extensive production systems in sub-tropical conditions.