Progesterone-based hormonal protocols are effective to treat anestrus in buffaloes. However, most of those protocols are costly, thus, requiring a cost-effective protocol that minimizes handling frequencies. This study, therefore, evaluated the effectiveness of the ‘once-used CIDR co-synch protocol’ with or without initial GnRH for treating anestrus in buffaloes during low breeding season. Anestrous buffaloes (n=29) were divided into two Treatment groups. As a part of the ‘once-used CIDR co-synch protocol,’ the first group (T1, n=18) received an initial GnRH on a random day (day 0) while the second group (T2, n=11) did not receive it. On day 0, the once-used CIDR device was inserted intravaginally in both groups, with GnRH administered in T1 but omitted in T2. On day 7, during CIDR removal, each buffalo received PGF2α, and on day 10, GnRH was given, and fixed-timed artificial insemination (FTAI) was done. Ovarian status was assessed on d0 and on 8 to 9-day post-FTAI using transrectal ultrasonography. Plasma progesterone concentrations on 8 to 9-day post-FTAI were determined using ELISA. No significant differences in estrus expression, ovulation and pregnancy rates were observed between two Treatment groups, with overall rates of 93%, 69%, and 34.5%, respectively. However, the overall pregnancy outcome (from FTAI plus natural breeding of buffaloes that returned to estrus) up to 2 months post-FTAI was 48.3%. Parity affected the ovulation among treated buffaloes, with higher (P=0.04) ovulation rate in buffaloes ≤4th parity (79.2%) compared to >4th parity (20%). There were no differences in CL size and plasma progesterone concentrations during 8 to 9-day post-FTAI between two treatment groups. In conclusion, omitting the initial GnRH in ‘once-used CIDR Co-synch protocol’ proved equally effective as the protocol with initial GnRH for the treatment of anestrus in buffaloes during the low breeding season.