Heat stress and essential amino acid profiles are important factors that can affect milk fat and protein synthesis in the mammary gland of dairy cows. MicroRNAs (miRNA) are non-coding RNAs that play a critical role in post-transcriptional regulation of bovine mammary gene expression. Regulatory microRNAs affect a wide variety of cellular processes such as cellular differentiation, proliferation, apoptosis, genome stability and milk protein and fat synthesis pathways. Although miRNA expression profiling in various bovine tissues or cells have been performed, studies to elucidate the potential role of miRNAs under stress conditions are limited or nonexistent. The objective of this study was to determine using MACT cells the effects of heat stress and amino acid (AA) supplementation on microRNA expression. A thermo neutral (TN, 37°C) and heat stress (HS, 42°C) environment with one of 3 AA supplementation groups were studied: control (Con), control + methionine (Met) and control + arginine (Arg). A total of 6 treatments were evaluated: TN-Con, TN-Met, TN-Arg, HS-Con, HS-Met and HS-Arg. After 6 h of incubation cells were harvested, and miRNAs were extracted for quantitative RT-PCR analysis. Targets included miR-141, miR-221, miR-34a, miR-200a, miR-27a/b, miR-92a, miR-99a, miR-23a, miR-26a, miR-103, and miR-184. Both HS and AA increased (P < 0.05) the expression of miR-141 and miR-221, which were reported to decrease STAT5 protein expression and induce angiogenesis. An upregulation (P < 0.05) of miR-34a, miR-200a, miR-27a/b, miR-92a, miR-99a, and miR-184 was observed in the HS group, but not with AA supplementation. These miRNAs are reported to be involved in the regulation of heat-shock proteins, milk fat synthesis, prolactin receptor expression, cell cycle regulation, and mTOR and insulin signaling. Both miR-23a (MAPK pathway) and miR-26a (cell proliferation) were not affected (P > 0.05) by temperature, but Met and/or Arg decreased (P < 0.05) their expression. The sole miRNA unaffected by temperature or AA was miR-103. The results suggest that AA supplementation and HS can alter miRNA expression and, as such, could serve as another regulatory factor of genes that play important functions in the mammary gland.