What one change to your room, system, or setup made the biggest improvement?

For me I think that the most important thing has been synergy between components of my system. The electronics are all McIntosh, and speakers in the 5.2 system are all Legacy Audio. The Mac and Legacy stuff go great together. 

Where to start? On the analog side, when I moved to a line stage preamp, I had to get a phone stage.  Incredible improvement.  On the digital side, there are a couple of things that really moved the needle.  The first was a fiber media conversion on the ethernet running into my streamer, best bang for the buck.  Getting a power conditioner and upgrading my power cords were also massive leaps forward.  Aiming for room treatments this summer.

Only problem was that the new room sounded horrible.

What finally changed it for me was purchasing two corner bass traps from ATS Acoustics and placed them on the front wall corners behind the speakers. Best $600-700 I ever spent. There was an immediate and profound difference in what I was hearing. Vocals were clearer and bass was smoother and more even. I was now able to listen for a few hours at a time whereas before, I would get listener fatigue after about 45-60 minutes.

Since the bass traps did wonders, I picked up matching absorption 2'x4' panels for the side walls along with 3D printed diffuser panels I picked up from an Audiogon listing. All of this made an amazing transformation and didn't cost an insane amount of money or require a lot of effort to install. Best part about this is, that it works with any equipment upgrade I may make in the future. Wish I had done this sooner.
 

@bflopez +1: this is almost exactly the same steps and experience I had with ATS corner bass traps and matching absorption/diffusion. Thank you for validating my journey with treatments! 

My Cayin 50 tube amp....I love it....so warm sounding.

Hard to assign true impact differentials, since all these upgrades work cumulatively. Once I settled on my components, I was impressed by the improvements from:
- Room treatment (corner and wall bass traps)
- Fidelium speaker cables and interconnects 
- Townshend speaker podia
- Dedicated power + SR power conditioner 

They all helped with better definition, separation, depth, timing.