This study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5–8 mm, third group 9–15 mm and fourth group 16–20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5–8 mm, 9–15 mm and 16–20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.