Microalgae are promising natural resources for an array of essential nutrients and health-promoting compounds. The past decade has witnessed their increasing popularity as a value-added ingredient in the food industry. However, some of their intrinsic properties, especially their typical green color and off-flavor, may compromise consumer acceptability and remain a key challenge for food scientists. This study aimed to generate and characterize a chemically induced pigment-deficient mutant (C49) of Chlorella pyrenoidosa. In contrast to the wild type (WT), C49 could not grow photoautotrophically or mixotrophically. However, when grown heterotrophically, it exhibited a growth curve similar to that of WT. Pigment analysis verified the presence of only trace amounts of chlorophylls (0.27 ± 0.02 mg/g) and carotenoids (0.036 ± 0.003 mg/g) in the C49 which were only ∼1.7% and ∼1% of those in the WT, respectively. C49 also had higher carbohydrate (62.75% ± 2.42%) and lower protein (26.54% ± 1.00%) content than the WT (37.41% ± 2.25% and 36.21% ± 1.97%, respectively). Genetic assays revealed inactivation of the phytoene desaturase gene as a key mechanism underlying the reduced xanthophyll-synthesizing capability of C49, thus causing its failure to accumulate chlorophyll and develop chloroplasts. Transcriptomic and RT-PCR analyses indicated that the high carbohydrate content was likely due to reduced starch degradation. Furthermore, C49 demonstrated an emulsifying capacity comparable to that of WT over a wide pH range (3–8) in oil-in-water emulsions consisting of 5% cell-disrupted homogenate in deionized water and rapeseed oil (1:1, v/v). The results of this study support that depigmented Chlorella pyrenoidosa possesses promising potential for application in the food industry.