Abstract Adipose tissue deposition in cattle can prove advantageous and detrimental when grading carcasses. Intramuscular fat (IMF) deposition, or marbling, can enhance sensory qualities such as tenderness and flavor, whereas subcutaneous fat deposition reduces carcass red meat yield. Little is known about how different mixtures of fatty acids influence adipocyte hypertrophy in vitro. Primary preadipocytes were isolated from subcutaneous fat harvested from Angus-cross yearling steers. Preadipocytes were propagated and differentiated into adipocytes. Then eight different mixtures with pairs of different fatty acids (C16:0, C18:0, C18:1cis-9, and C18:2) at varying ratios were added to the cultures. Each individual mix was applied at the same total molar concentration. At the end of the 4-day treatment period, cells were harvested for future RNA extraction and fatty acid analysis. The expression of critical adipogenic and lipogenic genes was evaluated. Mixes exhibited differences in total fatty acid content (ug) which would not only confirm that the adipocytes are differentiated but also translates to potential differences in hypertrophic growth between mixes (P < 0.05). The control exhibited significantly different fatty acid binding protein-4 (FABP4) and stearoyl co-A desaturase-1 (SCD-1) expressions in comparison with all other mixes (P < 0.05). The expression of FABP4 in mix 3 (75% C16:0-25% C18:1cis-9) was substantially different from all other treatments aside from a similarly structured mix (50% C16:0-50% C18:1cis-9; P < 0.05). The SCD-1 was upregulated, while peroxisome proliferating-activated receptor gamma (PPARy) was downregulated in the fully saturated fatty acid mix, mix 5 (50% C16:0- 50% C18:0), compared with all other treatments (P < 0.05). All three mixes containing C18:2cis-9 exhibited a greater relative expression of sterol-regulatory element-binding protein (SREB) than all other mixes (P < 0.05). The supplementation of fatty acids at different ratios can affect molecular mechanisms and hypertrophic growth in differentiated bovine adipocytes. Further investigation of this type of fatty acid supplementation could impact the future understanding of hypertrophic growth in the bovine adipocyte and provide further insight into the alteration of fat deposition in cattle.