Gloucester, who finished top of Pool 1, a point ahead of their French rivals, won the first meeting 35-14 at Kingsholm, before La Rochelle got their revenge with a convincing 42-13 victory on home soil.
Now the Cherry and Whites face another trip to France for a game with a La Rochelle team that has won 14 of their last 15 games. Patrice Collazo's side are 10 points clear at the Top14's summit and remain in contention for what would be a shock domestic and European double.
Gloucester need no reminding of how tough a challenge La Rochelle will provide.
"We've known this game has been coming for a while now," said Gloucester director of rugby Humphreys.
"It's a huge game and you want to be involved in these European semi-finals at this stage of the season.
"We also know the huge challenge we face going to La Rochelle having played them twice this year.
"There is a lot of expectation on them as they are the clear leaders in the Top14 which sums up the quality of the squad they have. They've rotated throughout the competition but no matter what team they put out, they will be unbelievably competitive.
"That's why for us, we have been looking at what we want and need to do when we approach the game, not just in the style we want to play, but to get mentally ready for a semi-final."
La Rochelle have been the surprise package in the Top14 - they lead second-placed Clermont by 10 points.
Gloucester, Challenge Cup winners in 2015, welcome back club captain Greig Laidlaw, who suffered ligament damage in the Six Nations.
There is less good news on centres Matt Scott and Andy Symons, winger Henry Purdy, and back-row Gareth Evans, who have all been ruled out.
La Rochelle's success is built on their power game with giant French forward Uini Atonio at the heart of their pack. Atonio's international team-mate Kevin Gourdon is also a key man.
La Rochelle coach Pierre Mignoni said: "We do not plan on the final stages. Our goal is to remain unbeaten at home.
"The group does not give up. La Rochelle deserves its course and we deserve to win one or two titles this season."
- This is Gloucester's fourth semi-final appearance in the Challenge Cup, and they've won their last two matches at this stage (L1). La Rochelle are in the last four for the first time.
- Gloucester's three previous semi-finals have all been against Aviva Premiership opposition, and their most recent success was against the Exeter Chiefs on their way to lifting the trophy in 2015.
- This will be the ninth meeting between the clubs in the Challenge Cup, and only Newcastle and Brive (12 times) have faced each other more often. Gloucester have won six of the previous eight meetings between the two (L2).
- Saturday's opponents have already met this season in the pool stage with the home side on the day winning each time. Both games were won by margins of more than 20 points.
- The Cherry and Whites have won seven of their last eight games away against Top 14 opposition, with their one defeat in that run coming against La Rochelle in Round 4.
- La Rochelle have won just three of their last 13 matches against Premiership sides in the Challenge Cup (D1, L9), although two of those victories have come in their last two home games.
- Gloucester have recorded the best ruck success in the Challenge Cup, winning 98% and losing just 12 overall this season.
- La Rochelle (8.7) are the only side this season to average fewer than 10 missed tackles per game. The Top 14 leaders' tackle success rate of 91% is the best in the competition.
- La Rochelle have stolen 19 line-outs this season, more than any other side, with Jason Eatonwinning claiming seven. No other player has stolen more than four.
- Billy Burns (7) and Billy Twelvetrees (6) have made the most and second-most try assists respectively this season, while Willi Heinz and La Rochelle pair Brock James and Kini Murimurivalu are three of four players to have completed five try assists.