Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.