Except for its use during milk protein synthesis, extra arginine (Arg) might exert other unknown biological or metabolic functions in the mammary gland. Previous work focused on the effect of Arg on bovine mammary epithelial cell casein production showed that 556.00 mg/L Arg (2 × the basal level) in the medium elicited the greatest stimulation of casein and mTOR-related genes. Whether Arg regulates cell development via effects on microRNA (miR) is unclear. The specific objective was to determine if miR expression in rat mammary tissue are altered by feeding 2 × Arg. Twelve pregnant littermate Wistar rats were randomly divided into 2 groups and experimental diets designed according to the AIN-93G purified diets. The Arg concentration was 12.8 g/kg in the Arg group while in the Control was 6.4 g/kg. A total of 21.62 g/kg glutamic acid was added to the Control group to keep diets iso-nitrogenous. At 17 d postpartum rats were killed and the mammary tissue harvested. Histological changes were measured by paraffin section, and mammary acinar area measured using digital imaging system software (MC30). Statistical analysis was carried out via t-test using SPSS16.0. Differentially expressed miR were measured using Solexa miR-Seq and verified by RT-qPCR. The miR target genes were analyzed using SBC prediction wizard (TargetScan, miRanda, PicTar, MirTarget2, PITA supported). Gene functions were analyzed via DAVID 6.7 and GO analysis. The mammary acinar area was significantly greater (P < 0.001) due to Arg (452.71 um²) compared with the Control (388.68 μm²). A total of 8 miR had greater (P < 0.05, fold-change > 2) expression in Arg compared with the Control. Among those upregulated, miR-1-3p plays a role in the control of cellular component synthesis; miR-133a-3p and miR-133a-5p play a role in the regulation of cell developmental process; miR-133b-3p exerts control of transporter activity and enzyme activity; and miR-206-3p has an important role in the regulation of cellular processes. There was no clear cellular function prediction for the upregulated miR such as miR-149-5p, miR-1b and miR-486. Results suggest dietary Arg might promote mammary tissue development through altering miR expression. The exact regulatory mechanisms need further investigation.