Background: Laparoscopic resection is increasingly used in colorectal cancer (CRC). It has been suggested to carry short-term benefits in safety, recovery, and preservation on immune function for patients with CRC. However, the impact of laparoscopic resection on natural killer (NK) cells is largely unclear. Methods: A total of 200 patients with CRC across Dukes A/B/C stages were randomly assigned to laparoscopic or open resection. The blood samples were collected before and after the surgery. The total number of NK cells was quantified by flow cytometer. Lytic units 35 toward K562 was used to quantify NK cells activity. The outcomes between the groups across pathological stages were also analyzed. Results: The number and activity of NK cells decreased after the surgery in both groups. The laparoscopic group showed a faster recovery rate of NK cells function than the control group as assessed by cell count and lytic activity. Natural killer cells were impaired in a higher degree in patients at Dukes B/C stages. The recovery of NK cells to baseline level at day 7 postsurgery was observed in the laparoscopic group across all 3 stages. Conclusion: Generally, laparoscopically assisted surgery resulted in a better preservation on NK cells function. A better outcome was observed in patients with CRC at Dukes B/C stages.