Granulosa cells (GCs) play a crucial role in follicular development and atresia. Previous studies have showed that GCs in the form of monolayer influenced in vitro maturation (IVM) of oocytes. However, the effects of GCs in the form of conditioned medium and monolayer on IVM and development competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of GC-conditioned medium (GCCM) and monolayer GC on maturation efficiency and embryo development of buffalo oocytes after parthenogenetic activation (PA). Our results showed that GCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2days and 20%) exerted significant negative effects on IVM rate (41.6% vs. 44.5%), but significantly enhanced embryo development (oocyte cleavage, 81.3% vs. 69.3%; blastocyst formation, 36.3% vs. 29.3%) of buffalo oocytes after PA compared with the control group. Furthermore, monolayer GC significantly reduced both maturation efficiency (40.2% vs. 44.5%) and embryo development (oocyte cleavage, 60.6% vs. 69.3%; blastocyst formation, 20.6% vs. 29.3%) of buffalo oocytes after PA compared to the control group. Our study indicated that GCs in the form of GCCM (2days and 20%) and monolayer GC had different effects on IVM and subsequent parthenogenetic development of buffalo oocytes.