You can play MP3 audio files during Twilio voice calls using the TwiML `<Play>` verb in Node.js. To do this, you'll need to set up a Node.js application with Twilio credentials, host an MP3 file either through Twilio Assets or your own Express server, and use the TwiML `<Play>` verb in your Twilio Programmable Voice API calls. You can play MP3 files on both incoming and outgoing voice calls using separate code paths, and you can also add additional functionality such as playing DTMF tones or looping audio files. The process involves setting up a local Express server to host the MP3 file, creating an ngrok tunnel to make the server publicly accessible, and then configuring your Twilio number to use the forwarding URL to play the MP3 file during incoming calls, or using a different code path for outgoing calls.