This study was conducted to investigate the effects of slaughter weight on chemical composition, physical properties, and fatty acid profile of musculus longissimus dorsi (MLD) in Holstein bulls. The bulls (n = 20) were divided into two slaughter weight groups as light (450-520 kg) and heavy (521-580 kg) according to body weights at slaughter. After resecting MLD from the carcass, its chemical composition, physical properties, and fatty acid profile were determined. The analysis showed that fat, ash, and cholesterol contents of MLD were significantly affected by slaughter weight (P < 0.05). Higher fat, ash, and cholesterol contents were determined in heavy bulls compared with light ones. However, the protein and moisture contents were not significantly different between slaughter weight groups in terms of pH, drip loss, and water holding capacity. With regard to meat color, the a* was significantly higher in light bulls; however, the L* and b* values were similar in both slaughter weight groups. Of all fatty acids, only C:10 was found the highest in heavy bulls (P < 0.05). The n-6/n-3 ratio was significantly lower (P < 0.05) in light bulls compared with heavy ones. Consequently, the best results were obtained from light Holstein bulls in order to achieve better beef quality.